Methods and Monitoring of Treatment With A DLL4 Antagonist

ABSTRACT

Methods for treating diseases such as cancer comprising administering a DLL4 antagonist, either alone or in combination with other anti-cancer agents, and monitoring for cardiovascular side effects and/or toxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/068,910, filed Oct. 31, 2013, which claims priority benefit of U.S.Provisional Application No. 61/720,768, filed Oct. 31, 2012 which ishereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2293_0980004_SEQLISTING.ascii.txt; Size: 17,185 bytes; and Date ofCreation: Feb. 1, 2017) is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

The present invention relates to the field of treating diseases with aDLL4 antagonist. More particularly, the invention provides methods fortreating cancer comprising administering a DLL4 antagonist, either aloneor in combination with other anti-cancer agents, and monitoring forcardiovascular side effects and/or toxicity.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich—breast, lung, colorectal, and prostate—account for almost half ofall new cases (Siegel et al., 2011, CA: A Cancer J. Clin. 61:212-236).

The Notch pathway is involved in multiple aspects of vasculardevelopment including proliferation, migration, smooth muscledifferentiation, angiogenesis, and arterial-venous differentiation (Isoet al., 2003, Arterioscler. Thromb. Vasc. Biol., 23:543). The Notchreceptor ligand DLL4 (Delta-like ligand 4) is an important component ofthe Notch pathway and plays a role in angiogenesis. Heterozygous loss ofDLL4 results in severe defects in arterial development and yolk sacvascularization, leading to embryonic lethality (Duarte et al., 2004,Genes Dev., 18:2474-78; Gale et al., 2004, PNAS, 101:15949-54; Krebs etal., 2004, Genes Dev., 18:2469-73). Furthermore, tumor cells and tumorvasculature often over-express DLL4, suggesting that DLL4 expression isan important player in tumor angiogenesis (Patel et al., 2005, CancerRes., 65:8690-97; Yan et al., 2001, Blood, 98:3793-99). Thus, blockingDLL4 signaling has emerged as a promising path for the development ofnew anti-cancer therapies.

Blocking DLL4 signaling, such as by an anti-DLL4 antibody, has beenshown to reduce tumor growth by multiple different mechanisms (Ridgwayet al., 2006, Nature, 444:1083-87; Noguera-Troise et al., Nature,444:1032-37; Hoey et al., 2009, Cell Stem Cell, 5:168-77). For example,DLL4 blocking antibodies have been reported to result in endothelialcell proliferation and the development of blood vessels, however, theseblood vessels lack a functional lumen. This dysangiogenic effect hasbeen reported to block tumor growth by promoting the development of onlynon-functional blood vessels (Ridgway et al., 2006, Nature, 444:1083-87;Noguera-Troise et al., Nature, 444:1032-37; Scehnet et al., 2007, Blood,109:4753-60). Additionally, DLL4 blocking antibodies have been shown toinhibit tumor growth by reducing the proliferation of tumor cells andreducing cancer stem cell frequency. Although the mechanism behind thereduction of cancer stem cells or CSCs is unknown, it is hypothesizedthat DLL4 is required for the self-renewal of CSCs and maintains thesecells in an undifferentiated state (Hoey et al., 2009, Cell Stem Cell,5:168-77).

Unlike therapeutic approaches that attempt to block the signaling oftumor angiogenic factors, blockade of DLL4 signaling by anti-human DLL4antibodies can result in endothelial hypertrophy and the creation ofnon-functional microvessels. Consequently, even in the presence of tumorangiogenic factors, blockade of DLL4 signaling, through administrationof anti-human DLL4 antibodies, can result in dysangiogenesis whichinhibits the ability of the tumor to induce the functional blood vesselformation needed to support growth of the tumor.

Chemotherapy is a well-established therapeutic approach for numerouscancers, but its efficacy can be limited by side effects and/ortoxicity. In addition, targeted therapies such as the anti-ErbB2receptor (HER2) antibody trastuzumab (HERCEPTIN), tyrosine kinaseinhibitors imatinib (GLEEVEC), dasatinib (SPRYCEL), nilotibib (TASIGNA),sunitinib (SUTENT), sorafenib (NEXAVAR), the anti-VEGF antibodybevacizumab (AVASTIN), and anti-angiogenesis drugs sunitinib (SUTENT)and sorafenib (NEXAVAR), are known to cause, or are likely to cause,side effects and/or toxicity in subjects who take them. For example,bevacizumab, sunitinib, and sorfenib are known to cause hypertension inabout one-third of patients who take them. In recent studies it has beenfound that anti-DLL4 antibodies may have side effects and/or toxicity insome subjects. For example, it has been found that anti-DLL4 antibodiescan cause hypertension in some patients. This was surprising, sinceanti-DLL4 antibodies have been reported to inhibit tumor angiogenesis bypromoting dysangiogensis, a mechanism different than that of traditionalanti-angiogenic treatments.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved methods for treating diseasescomprising administering to a subject a therapeutically effective amountof a DLL4 antagonist. For example, in one aspect the invention providesmethods of screening for, detecting, identifying, monitoring, reducing,preventing, and/or attenuating a cardiovascular side effect and/ortoxicity related to treatment with a DLL4 antagonist. In someembodiments, the methods comprise determining the level of a natriureticpeptide in a sample from a patient who has received, is receiving, willreceive, or is being considered for initial or further treatment with aDLL4 antagonist, including but not limited to an anti-DLL4 antibody.

In another aspect, the invention provides methods of selecting a subjectfor treatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject, determining the level of a biomarker in thesample, and selecting the subject for treatment with the DLL4 antagonistif the level of the biomarker is below a predetermined level. In someembodiments, the biomarker is a natriuretic peptide. Thus, in someembodiments, the method of selecting a subject for treatment with a DLL4antagonist, comprises: obtaining a biological sample from the subject,determining the level of a natriuretic peptide in the sample, andselecting the subject for treatment with the DLL4 antagonist if thelevel of the natriuretic peptide is below a predetermined level. In someembodiments, the biological sample is blood, serum, or plasma. In someembodiments, the natriuretic peptide is B-type natriuretic peptide(BNP). In some embodiments, the predetermined level is about 300 pg/mlor less in a blood, serum, or plasma sample. In some embodiments, thepredetermined level is about 200 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 250pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 200 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 150pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 100 pg/ml or less in a blood, serum, orplasma sample.

In another aspect, the invention provides methods of identifying asubject as eligible for treatment with a DLL4 antagonist, comprising:obtaining a biological sample from the subject, determining the level ofa biomarker in the sample, and identifying the subject as eligible fortreatment with the DLL4 antagonist if the level of the biomarker isbelow a predetermined level. In some embodiments, the biomarker is anatriuretic peptide. In some embodiments, the method of identifying asubject as eligible for treatment with a DLL4 antagonist, comprises:obtaining a biological sample from the subject, determining the level ofa natriuretic peptide in the sample, and identifying the subject aseligible for treatment with the DLL4 antagonist if the level of thenatriuretic peptide is below a predetermined level. In some embodiments,the natriuretic peptide is BNP. In some embodiments, the biologicalsample is blood, serum, or plasma. In some embodiments, thepredetermined level is about 300 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 200pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 250 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 200pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 150 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 100pg/ml or less in a blood, serum, or plasma sample.

In another aspect, the invention provides methods of monitoring asubject receiving treatment with a DLL4 antagonist for the developmentof cardiovascular side effects and/or toxicity, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof cardiovascular side effects and/or toxicity. In some embodiments, thebiomarker is a natriuretic peptide. In some embodiments, the method ofmonitoring a subject receiving treatment with a DLL4 antagonist for thedevelopment of cardiovascular side effects and/or toxicity, comprises:obtaining a biological sample from the subject receiving treatment,determining the level of a natriuretic peptide in the sample, andcomparing the level of the natriuretic peptide in the sample to apredetermined level of the natriuretic peptide, wherein an increase inthe level of the natriuretic peptide indicates development ofcardiovascular side effects and/or toxicity. In some embodiments, thenatriuretic peptide is BNP.

In another aspect, the invention provides methods of detecting thedevelopment of cardiovascular side effects and/or toxicity in a subjectreceiving treatment with a DLL4 antagonist, comprising: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof cardiovascular side effects and/or toxicity. In some embodiments, thebiomarker is a natriuretic peptide. In some embodiments, the method ofdetecting the development of a cardiovascular side effect and/ortoxicity in a subject receiving treatment with a DLL4 antagonist,comprises: obtaining a biological sample from the subject receivingtreatment, determining the level of a natriuretic peptide in the sample,and comparing the level of the natriuretic peptide in the sample to apredetermined level of the natriuretic peptide, wherein an increase inthe level of the natriuretic peptide indicates development of acardiovascular side effect and/or toxicity. In some embodiments, thenatriuretic peptide is BNP.

In another aspect, the invention provides methods for identifyingcardiovascular side effects and/or toxicity in a subject receivingtreatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject receiving treatment, determining the level of abiomarker in the sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then a cardiovascular side effect and/or toxicity isindicated. In some embodiments, the biomarker is a natriuretic peptide.In some embodiments, the method for identifying cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist, comprises: obtaining a biological sample from the subjectreceiving treatment, determining the level of a natriuretic peptide inthe sample, and comparing the level of the natriuretic peptide in thesample to a predetermined level of the natriuretic peptide, wherein ifthe level of the natriuretic peptide in the sample is higher than thepredetermined level of the natriuretic peptide then a cardiovascularside effect and/or toxicity is indicated. In some embodiments, thenatriuretic peptide is BNP.

In another aspect, the invention provides methods for monitoringcardiovascular side effects and/or toxicity in a subject receivingtreatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject receiving treatment, determining the level of abiomarker in the sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then a cardiovascular side effect and/or toxicity isindicated. In some embodiments, the biomarker is a natriuretic peptide.In some embodiments, the method for monitoring cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist, comprises: obtaining a biological sample from the subjectreceiving treatment, determining the level of a natriuretic peptide inthe sample, and comparing the level of the natriuretic peptide in thesample to a predetermined level of the natriuretic peptide, wherein ifthe level of the natriuretic peptide in the sample is higher than thepredetermined level of the natriuretic peptide then a cardiovascularside effect and/or toxicity is indicated. In some embodiments, thenatriuretic peptide is BNP.

In some aspects and/or embodiments of the methods described herein, thebiological sample is blood, serum, or plasma. In some embodiments, thepredetermined level is about 300 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 200pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 250 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 200pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level is about 150 pg/ml or less in a blood, serum, orplasma sample. In some embodiments, the predetermined level is about 100pg/ml or less in a blood, serum, or plasma sample. In some embodiments,the predetermined level of a biomarker (e.g., natriuretic peptide orBNP) is the amount of the biomarker in a sample obtained at an earlierdate. In some embodiments, the predetermined level of a biomarker (e.g.,natriuretic peptide or BNP) is the amount of the biomarker in a sampleobtained prior to treatment. In some embodiments, the predeterminedlevel of a biomarker (e.g., natriuretic peptide or BNP) is a normalreference level. In some embodiments, the predetermined level for BNP isabout 100 pg/ml or less in blood, serum, or plasma.

In some aspects and/or embodiments of the methods described herein, abiological sample is obtained approximately every week, every 2 weeks,every 3 weeks, or every 4 weeks.

In some aspects and/or embodiments of the methods described herein,wherein if the natriuretic peptide level in the sample is above apredetermined level for two consecutive samples, the subject isadministered a therapeutically effective amount of a cardioprotectivemedication such as an ACE inhibitor and/or a β-blocker. In someembodiments, the natriuretic peptide is BNP and the predetermined levelis about 100 pg/ml. In some embodiments of the methods described herein,wherein if the natriuretic peptide level in the sample is above apredetermined level for any one sample, the subject is administered atherapeutically effective amount of an inhibitor and/or a β-blocker. Insome embodiments, the natriuretic peptide is BNP and the predeterminedlevel is about 200 pg/ml. In some embodiments of the methods describedherein, wherein the natriuretic peptide level in the sample is above apredetermined level for any one sample, the subject is administered atherapeutically effective amount of an ACE inhibitor and/or a β-blockerand the DLL4 antagonist is withheld. In some embodiments, thenatriuretic peptide is BNP and the predetermined level is about 300pg/ml. In some embodiments, if the natriuretic peptide level decreasesto below about 200 pg/ml after administration of the ACE inhibitorand/or a β-blocker, then administration of the DLL4 antagonist isresumed. In some embodiments, if the natriuretic peptide level decreasesto below about 300 pg/ml after administration of the ACE inhibitorand/or a β-blocker, then administration of the DLL4 antagonist isresumed.

In another aspect, the invention provides methods of reducingcardiovascular side effects and/or toxicity in a subject receivingtreatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject receiving treatment, determining the level of anatriuretic peptide in the sample, comparing the level of thenatriuretic peptide in the sample to a predetermined level of thenatriuretic peptide, and administering to the subject a therapeuticallyeffective amount of a cardioprotective medication such as an ACEinhibitor and/or a β-blocker if the level of the natriuretic peptide inthe sample is higher than the predetermined level of the natriureticpeptide. In another aspect, the invention provides methods of preventingor attenuating the development of cardiovascular side effects and/ortoxicity in a subject receiving treatment with a DLL4 antagonist,comprising: obtaining a biological sample from the subject prior totreatment with the DLL4 antagonist, determining the level of anatriuretic peptide in the sample, comparing the level of thenatriuretic peptide in the sample to a predetermined level of thenatriuretic peptide, administering to the subject a therapeuticallyeffective amount of a cardioprotective medication such as an ACEinhibitor and/or a β-blocker, and administering to the subject the DLL4antagonist. In another aspect, the invention provides methods ofameliorating cardiovascular side effects and/or toxicity in a subjectadministered a DLL4 antagonist, comprising: determining the level of anatriuretic peptide in the sample, and administering to the subject atherapeutically effective amount of a cardioprotective medication suchas an ACE inhibitor and/or a β-blocker. In some embodiments, thenatriuretic peptide is BNP.

In another aspect, the invention provides methods of screening a subjectfor the risk of cardiovascular side effects and/or toxicity fromtreatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject prior to treatment with the DLL4 antagonist,determining the level of a natriuretic peptide in the sample, andcomparing the level of the natriuretic peptide in the sample to apredetermined level of the natriuretic peptide, wherein if the level ofthe natriuretic peptide in the sample is higher than the predeterminedlevel then the subject is at risk for cardiovascular side effects and/ortoxicity. In some embodiments, if the subject is at risk forcardiovascular side effects and/or toxicity, the subject is administereda therapeutically effective amount of a therapeutic agent directed tothe cardiovascular side effect and/or toxicity prior to treatment withthe DLL4 antagonist.

In any of the aspects and/or embodiments of the methods describedherein, the DLL4 antagonist specifically binds human DLL4. In someembodiments, the DLL4 antagonist is an antibody that specifically bindsthe extracellular domain of human DLL4. In some embodiments, the DLL4antagonist specifically binds an epitope within amino acids 27-217 ofthe extracellular domain of human DLL4 (SEQ ID NO: 17). In someembodiments, the DLL4 antagonist binds an epitope comprising amino acids66-73 (QAVVSPGP, SEQ ID NO: 18) of human DLL4. In some embodiments, theDLL4 antagonist binds an epitope comprising amino acids 139-146(LISKIAIQ, SEQ ID NO: 19) of human DLL4. In some embodiments, the DLL4antagonist binds an epitope comprising amino acids 66-73 (QAVVSPGP, SEQID NO: 18) and amino acids 139-146 (LISKIAIQ, SEQ ID NO: 19) of humanDLL4. In some embodiments, the DLL4 antagonist binds human DLL4 with adissociation constant (K_(D)) of about 10 nM to about 0.1 nM.

In certain embodiments, the DLL4 antagonist is an anti-DLL4 antibody. Incertain embodiments, the DLL4 antagonist is an antibody comprising aheavy chain CDR1 comprising TAYYIH (SEQ ID NO: 1), a heavy chain CDR2comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS(SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:8). In certain embodiments, the DLL4 antagonist is an antibodycomprising a heavy chain variable region comprising the amino acids ofSEQ ID NO: 10. In certain embodiments, the DLL4 antagonist is anantibody which further comprises a light chain variable regioncomprising the amino acids of SEQ ID NO: 12. In certain embodiments, theDLL4 antagonist comprises the same heavy and light chain amino acidsequences as an antibody encoded by a plasmid deposited with ATCC havingdeposit no. PTA-8425 or PTA-8427. In certain embodiments, the DLL4antagonist comprises the heavy chain CDR amino acid sequences and thelight chain CDR amino acid sequences that are contained in the 21M18antibody produced by the hybridoma deposited with ATCC having depositno. PTA-8670. In certain embodiments, the DLL4 antagonist is encoded bythe plasmid having ATCC deposit no. PTA-8425 which was deposited withAmerican Type Culture Collection (ATCC), at 10801 University Boulevard,Manassas, Va., 20110, under the conditions of the Budapest Treaty on May10, 2007. In certain embodiments, the DLL4 antagonist is encoded by theplasmid having ATCC deposit no. PTA-8427 which was deposited withAmerican Type Culture Collection (ATCC), at 10801 University Boulevard,Manassas, Va., 20110, under the conditions of the Budapest Treaty on May10, 2007. In some embodiments, the DLL4 antagonist is the antibodyproduced by the hybridoma having ATCC deposit no. PTA-8670 which wasdeposited with the ATCC under the conditions of the Budapest Treaty onSep. 28, 2007. In some embodiments, the DLL4 antagonist is a humanizedversion of the antibody produced by the hybridoma having ATCC depositno. PTA-8670. In certain embodiments, the DLL4 antagonist competes forspecific binding to human DLL4 with an antibody encoded by the plasmiddeposited with ATCC having deposit no. PTA-8425 or PTA-8427.

In any of the aspects and/or embodiments of the methods describedherein, the subject has cancer. In some embodiments, the cancer isselected from the group consisting of: lung cancer, pancreatic cancer,breast cancer, colon cancer, colorectal cancer, melanoma,gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer,liver cancer, endometrial cancer, kidney cancer, prostate cancer,thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, and head and neckcancer.

In any of the aspects and/or embodiments of the methods describedherein, the subject is treated with the DLL4 antagonist in combinationwith one or more additional anti-cancer agents.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The table is a summary of some of the subjects enrolled in thePhase 1b clinical trial for treatment of NSCLC with OMP-21M18 incombination with carboplatin and pemetrexed.

FIG. 2. Percent change in target tumor lesions in subjects enrolled inthe Phase 1b clinical trial for treatment of NSCLC with OMP-21M18 incombination with carboplatin and pemetrexed.

FIG. 3. Percent change in target tumor lesions in subjects enrolled inthe Phase 1b clinical trial for treatment of pancreatic cancer withOMP-21M18 in combination with gemcitabine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to treating diseases with a DLL4antagonist. More particularly, the invention provides methods fortreating cancer comprising administering a DLL4 antagonist, either aloneor in combination with other anti-cancer agents, and monitoring forcardiovascular side effects and/or toxicity, including those related tothe DLL4 antagonist.

The anti-DLL4 antibody OMP-21M18 was administered to subjects in a Phase1 single agent dose escalation trial. The data from this early trial, aswell as results from animal studies suggested that administration of aDLL4 antagonist such as an anti-DLL4 antibody may result incardiovascular side effects and/or toxicity in certain patients.Furthermore, the study showed that increased BNP levels may be an earlyindicator that a patient being treated with a DLL4 antagonist is at riskof developing cardiotoxicity, allowing for intervention withcardioprotective medications.

These results made it desirable to develop risk mitigation andmonitoring strategies for cardiovascular side effects and/or toxicitiesas described herein for subjects receiving treatment with a DLL4antagonist (e.g., an anti-DLL4 antibody) as a single agent or incombination with additional anti-cancer agents.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “antagonist” and “antagonistic” as used herein refer to anymolecule that partially or fully blocks, inhibits, reduces orneutralizes a biological activity of a target and/or signaling pathway(e.g., Notch signaling). The term “antagonist” is used herein to includeany molecule that partially or fully blocks, inhibits, reduces orneutralizes the activity of a protein (e.g., DLL4). As used herein theterm “DLL4 antagonist” refers to a molecule that partially or fullyblocks, inhibits, neutralizes, or interferes with the biologicalactivities of a DLL4 protein. This includes, but is not limited to,blocking, inhibiting, reducing, or interfering with DLL4/Notchinteractions, DLL4-induced Notch pathway signaling, and/or DLL4signaling. Suitable antagonist molecules specifically include, but arenot limited to, antagonist DLL4 antibodies or antibody fragments.

The terms “modulation” and “modulate” as used herein refer to a changeor an alteration in a biological activity. Modulation includes, but isnot limited to, stimulating or inhibiting an activity. Modulation may bean increase or a decrease in activity (e.g., a decrease in Notchsignaling), a change in binding characteristics, or any other change inthe biological, functional, or immunological properties associated withthe activity of a protein, pathway, or other biological point ofinterest.

The term “antibody” as used herein refers to an immunoglobulin moleculethat recognizes and specifically binds a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing, through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term encompasses intact polyclonal antibodies, intactmonoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2,and Fv fragments), single chain Fv (scFv) antibodies, multispecificantibodies such as bispecific antibodies generated from at least twointact antibodies, monospecific antibodies, monovalent antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site as long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well-known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules, including but not limited to, toxins andradioisotopes.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. “Antibody fragment” as used herein comprises anantigen-binding site or epitope-binding site.

The term “variable region” of an antibody refers to the variable regionof the antibody light chain, or the variable region of the antibodyheavy chain, either alone or in combination. The variable regions of theheavy and light chain each consist of four framework regions (FR)connected by three complementarity determining regions (CDRs), alsoknown as “hypervariable regions”. The CDRs in each chain are heldtogether in close proximity by the framework regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsites of the antibody. There are at least two techniques for determiningCDRs: (1) an approach based on cross-species sequence variability (i.e.,Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5thEdition, National Institutes of Health, Bethesda Md.), and (2) anapproach based on crystallographic studies of antigen-antibody complexes(Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition,combinations of these two approaches are sometimes used in the art todetermine CDRs.

The term “monoclonal antibody” as used herein refers to a homogenousantibody population involved in the highly specific recognition andbinding of a single antigenic determinant or epitope. This is incontrast to polyclonal antibodies that typically include a mixture ofdifferent antibodies directed against different antigenic determinants.The term “monoclonal antibody” encompasses both intact and full-lengthmonoclonal antibodies as well as antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteins comprisingan antibody portion, and any other modified immunoglobulin moleculecomprising an antigen recognition site (antigen-binding site).Furthermore, “monoclonal antibody” refers to such antibodies made by anynumber of techniques, including but not limited to, hybridomaproduction, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” as used herein refers to forms ofantibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-humansequences. Typically, humanized antibodies are human immunoglobulins inwhich residues of the CDRs are replaced by residues from the CDRs of anon-human species (e.g., mouse, rat, rabbit, or hamster) that have thedesired specificity, affinity, and/or binding capability (Jones et al.,1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, theFv framework region residues of a human immunoglobulin are replaced withthe corresponding residues in an antibody from a non-human species thathas the desired specificity, affinity, and/or binding capability. Thehumanized antibody can be further modified by the substitution ofadditional residues either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or binding capability. In some embodiments, the humanizedantibody will comprise substantially all of at least one, and typicallytwo or three, variable domains containing all or substantially all ofthe CDRs that correspond to the non-human immunoglobulin whereas all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody can alsocomprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Examples ofmethods used to generate humanized antibodies are described in, forexample, U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein refers to an antibody producedby a human or an antibody having an amino acid sequence corresponding toan antibody produced by a human made using any of the techniques knownin the art. This definition of a human antibody specifically excludes ahumanized antibody comprising non-human antigen-binding residues.

The term “chimeric antibody” as used herein refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. Typically, the variable region of bothlight and heavy chains corresponds to the variable region of antibodiesderived from one species of mammals (e.g., mouse, rat, rabbit, etc.)with the desired specificity, affinity, and/or binding capability, whilethe constant regions are homologous to the sequences in antibodiesderived from another species (e.g., human).

The phrase “affinity-matured antibody” as used herein refers to anantibody with one or more alterations in one or more CDRs thereof thatresult in an improvement in the affinity of the antibody for antigen,compared to a parent antibody that does not possess thosealterations(s). Preferred affinity-matured antibodies will havenanomolar or even picomolar affinities for the target antigen.Affinity-matured antibodies are produced by procedures known in the art.For example, Marks et al., 1992, Bio/Technology 10:779-783, describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by Barbas et al., 1994, PNAS,91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al.,1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol.,154:3310-9; and Hawkins et al., 1992, J. Mol. Biol., 226:889-896.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids (also referredto as linear epitopes) are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding (also referred to asconformational epitopes) are typically lost upon protein denaturing. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation.

The terms “selectively binds” or “specifically binds” mean that abinding agent or an antibody reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein, or target moleculethan with alternative substances, including unrelated or relatedproteins. In certain embodiments “specifically binds” means, forinstance, that an antibody binds a protein with a K_(D) of about 0.1 mMor less, but more usually less than about 1 μM. In certain embodiments,“specifically binds” means that an antibody binds a target at times witha K_(D) of at least about 0.1 μM or less, at other times at least about0.01 μM or less, and at other times at least about 1 nM or less. Becauseof the sequence identity between homologous proteins in differentspecies, specific binding can include an antibody that recognizes aprotein in more than one species (e.g., human DLL4 and mouse DLL4).Likewise, because of homology within certain regions of polypeptidesequences of different proteins, specific binding can include anantibody (or other polypeptide or binding agent) that recognizes morethan one protein. It is understood that, in certain embodiments, anantibody or binding moiety that specifically binds a first target may ormay not specifically bind a second target. As such, “specific binding”does not necessarily require (although it can include) exclusivebinding, i.e. binding to a single target. Thus, an antibody may, incertain embodiments, specifically bind more than one target. In certainembodiments, multiple targets may be bound by the same antigen-bindingsite on the antibody. For example, an antibody may, in certaininstances, comprise two identical antigen-binding sites, each of whichspecifically binds the same epitope on two or more proteins. In someembodiments, an antibody may be bispecific or multispecific and compriseat least two antigen-binding sites with differing specificities. Forexample, a bispecific antibody may comprise one antigen-binding sitethat recognizes an epitope on one protein and further comprise a second,different antigen-binding site that recognizes a different epitope on asecond protein. Generally, but not necessarily, reference to bindingmeans specific binding.

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because the polypeptides of this invention may be basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains (e.g., dimers).

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to polymers of nucleotides of any length, and includeDNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known in the art. These include,but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. In some embodiments, two nucleic acids orpolypeptides of the invention are substantially identical, meaning theyhave at least 70%, at least 75%, at least 80%, at least 85%, at least90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm or byvisual inspection. In some embodiments, identity exists over a region ofthe sequences that is at least about 10, at least about 20, at leastabout 40-60 residues, at least about 60-80 residues in length or anyintegral value therebetween. In some embodiments, identity exists over alonger region than 60-80 residues, such as at least about 80-100residues, and in some embodiments the sequences are substantiallyidentical over the full length of the sequences being compared, such asthe coding region of a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s), i.e., the one or more RSPO protein(s) to which thepolypeptide or antibody binds. Methods of identifying nucleotide andamino acid conservative substitutions which do not eliminate antigenbinding are well-known in the art.

The term “vector” as used herein means a construct, which is capable ofdelivering, and usually expressing, one or more gene(s) or sequence(s)of interest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid, or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells, orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, a polypeptide, antibody, polynucleotide, vector, cell, orcomposition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, blastoma, sarcoma, andhematologic cancers such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass oftissue that results from excessive cell growth or proliferation, eitherbenign (non-cancerous) or malignant (cancerous) including pre-cancerouslesions.

The term “metastasis” as used herein refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates (e.g., via thebloodstream or lymph) from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumorinitiating cell” are used interchangeably herein and refer to cells froma cancer or tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more types ofdifferentiated cell progeny wherein the differentiated cells havereduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties confer on the cancer stem cells the ability to form orestablish a tumor or cancer upon serial transplantation into animmunocompromised host (e.g., a mouse) compared to the majority of tumorcells that fail to form tumors. Cancer stem cells undergo self-renewalversus differentiation in a chaotic manner to form tumors with abnormalcell types that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total populationof cells derived from a cancer or tumor or pre-cancerous lesion,including both non-tumorigenic cells, which comprise the bulk of thecancer cell population, and tumorigenic stem cells (cancer stem cells).As used herein, the terms “cancer cell” or “tumor cell” will be modifiedby the term “non-tumorigenic” when referring solely to those cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional featuresof a cancer stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of arandom sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The term “pharmaceutically acceptable” refers to approved or approvableby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant”or “acceptable pharmaceutical carrier” refer to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone binding agent (e.g., an antibody) of the present disclosure, andwhich does not destroy the pharmacological activity thereof and isnontoxic when administered in doses sufficient to deliver a therapeuticeffect.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a binding agent, an antibody,polypeptide, polynucleotide, small organic molecule, or other drugeffective to “treat” a disease or disorder in a subject. In the case ofcancer, the therapeutically effective amount of a drug (e.g., anantibody) has a therapeutic effect and as such can reduce the number ofcancer cells; decrease tumorigenicity, tumorigenic frequency, ortumorigenic capacity; reduce the number or frequency of cancer stemcells; reduce the tumor size; reduce the cancer cell population; inhibitor stop cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibit andstop tumor or cancer cell metastasis; inhibit and stop tumor or cancercell growth; relieve to some extent one or more of the symptomsassociated with the cancer; reduce morbidity and mortality; improvequality of life; or a combination of such effects. To the extent theagent, for example an antibody, prevents growth and/or kills existingcancer cells, it can be referred to as cytostatic and/or cytotoxic.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. In some embodiments, a subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including the spread of cancer cells into soft tissueand bone; inhibition of or an absence of tumor or cancer cellmetastasis; inhibition or an absence of cancer growth; relief of one ormore symptoms associated with the specific cancer; reduced morbidity andmortality; improvement in quality of life; reduction in tumorigenicity;reduction in the number or frequency of cancer stem cells; or somecombination of effects.

As used in the present disclosure and claims, the singular forms “a”,“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.It is also understood that wherever embodiments are described hereinwith the language “consisting essentially of” otherwise analogousembodiments described in terms of “consisting of” are also provided.

As used herein, reference to “about” or “approximately” a value orparameter includes (and describes) embodiments that are directed to thatvalue or parameter. For example, description referring to “about X”includes description of “X”.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. DLL4 Antagonists

In certain embodiments, the DLL4 antagonist specifically binds theextracellular domain of human DLL4. In some embodiments, the DLL4antagonist is an antibody. In some embodiments, the DLL4 antagonist orantibody specifically binds an epitope within amino acids 27-217 of theextracellular domain of human DLL4 (SEQ ID NO: 17). In some embodiments,the DLL4 antagonist or antibody specifically binds an epitope formed bya combination of the N-terminal region of human DLL4 (SEQ ID NO: 14) andthe DSL region of human DLL4 (SEQ ID NO: 15). In some embodiments, theDLL4 antagonist or antibody specifically binds within the N-terminalregion of human DLL4 (SEQ ID NO: 14). In some embodiments, the DLL4antagonist or antibody binds an epitope comprising amino acids 66-73(QAVVSPGP; SEQ ID NO: 18) of human DLL4. In some embodiments, the DLL4antagonist or antibody binds an epitope comprising amino acids 139-146(LISKIAIQ; SEQ ID NO: 19) of human DLL4. In some embodiments, the DLL4antagonist or antibody binds an epitope comprising amino acids 66-73(QAVVSPGP; SEQ ID NO: 18) and amino acids 139-146 (LISKIAIQ; SEQ ID NO:19) of human DLL4.

In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds tohuman DLL4 with a dissociation constant (K_(D)) of about 1 μM or less,about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10nM or less, or about 1 nM or less. In certain embodiments, the DLL4antagonist or antibody binds to human DLL4 with a K_(D) of about 40 nMor less, about 20 nM or less, about 10 nM or less, or about 1 nM orless. In certain embodiments, the DLL4 antagonist binds to human DLL4with a K_(D) of about 1 nM. In certain embodiments, the DLL4 antagonistbinds to human DLL4 with a K_(D) of about 0.8 nM. In certainembodiments, the DLL4 antagonist binds to human DLL4 with a K_(D) ofabout 0.6 nM. In certain embodiments, the DLL4 antagonist binds to humanDLL4 with a K_(D) of about 0.5 nM. In certain embodiments, the DLL4antagonist binds to human DLL4 with a K_(D) of about 0.4 nM. In someembodiments, the K_(D) is measured by surface plasmon resonance. In someembodiments, the dissociation constant of the antagonist or antibody toDLL4 is the dissociation constant determined using a DLL4 fusion proteincomprising a DLL4 extracellular domain (e.g., a DLL4 ECD-Fc fusionprotein) immobilized on a Biacore chip.

In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds toDLL4 with a half maximal effective concentration (EC₅₀) of about 1 μM orless, about 100 nM or less, about 40 nM or less, about 20 nM or less,about 10 nM or less, or about 1 nM or less. In certain embodiments, theDLL4 antagonist or antibody binds to human DLL4 with an EC₅₀ of about 40nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM orless.

In certain embodiments, the DLL4 antagonist is a polypeptide. In certainembodiments, the DLL4 antagonist or polypeptide is an antibody. Incertain embodiments, the antibody is an IgG antibody. In someembodiments, the antibody is an IgG1 antibody. In some embodiments, theantibody is an IgG2 antibody. In certain embodiments, the antibody is amonoclonal antibody. In some embodiments, the antibody is a bispecificantibody. In certain embodiments, the antibody is a humanized antibody.In certain embodiments, the antibody is a human antibody. In certainembodiments, the antibody is an antibody fragment comprising anantigen-binding site.

The DLL4 antagonists (e.g., antibodies) of the present invention can beassayed for specific binding by any method known in the art. Theimmunoassays which can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such asBiacore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA,“sandwich” immunoassay, immunoprecipitation assay, precipitationreaction, gel diffusion precipitin reaction, immunodiffusion assay,agglutination assay, complement-fixation assay, immunoradiometric assay,fluorescent immunoassay, and protein A immunoassay. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., Editors,1994-present, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y.).

In some embodiments, the specific binding of a DLL4 antagonist (e.g., anantibody) to human DLL4 may be determined using ELISA. An ELISA assaycomprises preparing DLL4 antigen, coating wells of a 96 well microtiterplate with antigen, adding to the wells the DLL4 antagonist or antibodyconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase), incubating for aperiod of time and detecting the presence of the binding agent orantibody. In some embodiments, the DLL4 antagonist or antibody is notconjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the DLL4 antagonist or antibody is added to thewell. In some embodiments, instead of coating the well with DLL4antigen, the DLL4 antagonist or antibody can be coated on the well,antigen is added to the coated well and then a second antibodyconjugated to a detectable compound is added. One of skill in the artwould be knowledgeable as to the parameters that can be modified and/oroptimized to increase the signal detected, as well as other variationsof ELISAs that can be used (see, e.g., Ausubel et al., Editors,1994-present, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y.).

In some embodiments, the specific binding of a DLL4 antagonist (e.g., anantibody) to human DLL4 may be determined using FACS. A FACS screeningassay may comprise generating a cDNA construct that expresses an antigenas a fusion protein, transfecting the construct into cells, expressingthe antigen on the surface of the cells, mixing the DLL4 antagonist withthe transfected cells, and incubating for a period of time. The cellsbound by the DLL4 antagonist may be identified by using a secondaryantibody conjugated to a detectable compound (e.g., PE-conjugatedanti-Fc antibody) and a flow cytometer. One of skill in the art would beknowledgeable as to the parameters that can be modified to optimize thesignal detected as well as other variations of FACS that may enhancescreening (e.g., screening for blocking antibodies).

The binding affinity of an antagonist or antibody to DLL4 and the on-offrate of an antibody-antigen interaction can be determined by competitivebinding assays. In some embodiments, a competitive binding assay is aradioimmunoassay comprising the incubation of labeled antigen (e.g., ³Hor ¹²⁵I), or fragment or variant thereof, with the antibody of interestin the presence of increasing amounts of unlabeled antigen followed bythe detection of the antibody bound to the labeled antigen. The affinityof the antibody for the antigen and the on-off rates can be determinedfrom the data by Scatchard plot analysis. In some embodiments, Biacorekinetic analysis is used to determine the binding affinities and on-offrates of antagonists or antibodies that bind DLL4. Biacore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom antigens (e.g., DLL4 proteins) that have been immobilized on thesurface of a Biacore chip. In some embodiments, Biacore kinetic analysescan be used to study binding of different antibodies in qualitativeepitope competition binding assays.

In some embodiments, the DLL4 antagonists are polyclonal antibodies.Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies are prepared by immunizing an animal (e.g., a rabbit, rat,mouse, goat, donkey) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (e.g., a purified peptide fragment,full-length recombinant protein, fusion protein, etc.). The antigen canbe optionally conjugated to a carrier protein such as keyhole limpethemocyanin (KLH) or serum albumin. The antigen (with or without acarrier protein) is diluted in sterile saline and usually combined withan adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form astable emulsion. After a sufficient period of time, polyclonalantibodies are recovered from blood, ascites, and the like, of theimmunized animal. Polyclonal antibodies can be purified from serum orascites according to standard methods in the art including, but notlimited to, affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In some embodiments, the DLL4 antagonists are monoclonal antibodies.Monoclonal antibodies can be prepared using hybridoma methods known toone of skill in the art (see e.g., Kohler and Milstein, 1975, Nature256:495). Using the hybridoma method, a mouse, hamster, or otherappropriate host animal, is immunized as described above to elicitlymphocytes to produce antibodies that will specifically bind theimmunizing antigen. In some embodiments, lymphocytes are immunized invitro. In some embodiments, the immunizing antigen (e.g., DLL4) is ahuman protein or a portion thereof. In some embodiments, the immunizingantigen (e.g., DLL4) is a mouse protein or a portion thereof. In someembodiments, the immunizing antigen is an extracellular domain of humanDLL4. In some embodiments, the immunizing antigen is an extracellulardomain of mouse DLL4. In some embodiments, a mouse is immunized with ahuman antigen. In some embodiments, a mouse is immunized with a mouseantigen.

Following immunization, lymphocytes are isolated and fused with asuitable myeloma cell line using, for example, polyethylene glycol. Thehybridoma cells are selected using specialized media as known in the artand unfused lymphocytes and myeloma cells do not survive the selectionprocess. Hybridomas that produce monoclonal antibodies directed againsta target antigen may be identified by a variety of techniques including,but not limited to, immunoprecipitation, immunoblotting, and in vitrobinding assays (e.g., flow cytometry, enzyme-linked immunosorbent assay(ELISA), or radioimmunoassay (RIA)). The hybridomas can be propagatedeither in in vitro culture using standard methods (J. W. Goding, 1996,Monoclonal Antibodies: Principles and Practice, 3rd Edition, AcademicPress, San Diego, Calif.) or in vivo as ascites in a host animal. Themonoclonal antibodies can be purified from the culture medium or ascitesfluid according to standard methods in the art including, but notlimited to, affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In some embodiments, monoclonal antibodies can be made using recombinantDNA techniques as known to one skilled in the art (see e.g., U.S. Pat.No. 4,816,567). For example, the polynucleotides encoding a monoclonalantibody are isolated from mature B-cells or hybridoma cells, such as byRT-PCR using oligonucleotide primers that specifically amplify the genesencoding the heavy and light chains of the antibody, and their sequenceis determined using conventional techniques. The isolatedpolynucleotides encoding the heavy and light chains are cloned intosuitable expression vectors which produce the monoclonal antibodies whentransfected into host cells such as E. coli, simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin proteins. In certain embodiments, recombinantmonoclonal antibodies, or fragments thereof, can be isolated from phagedisplay libraries expressing variable domain regions or CDRs of adesired species (see e.g., McCafferty et al., 1990, Nature, 348:552-554;Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J.Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can be modified,for example, by using recombinant DNA technology to generate alternativeantibodies. In some embodiments, the constant domains of the light andheavy chains of, for example, a mouse monoclonal antibody can besubstituted for those regions of, for example, a human antibody togenerate a chimeric antibody or for a non-immunoglobulin polypeptide togenerate a fusion antibody. In some embodiments, the constant regionsare truncated or removed to generate the desired antibody fragment of amonoclonal antibody. In some embodiments, site-directed or high-densitymutagenesis of the variable region can be used to optimize specificity,affinity, and/or other biological characteristics of a monoclonalantibody. In some embodiments, site-directed mutagenesis of the CDRs canbe used to optimize specificity, affinity, and/or other biologicalcharacteristics of a monoclonal antibody.

In some embodiments, the DLL4 antagonist is a humanized antibody.Typically, humanized antibodies are human immunoglobulins in whichresidues from the complementary determining regions (CDRs) are replacedby residues from CDRs of a non-human species (e.g., mouse, rat, rabbit,hamster) that have the desired specificity, affinity, and/or capabilityby methods known to one skilled in the art. In some embodiments, the Fvframework region residues of a human immunoglobulin are replaced withthe corresponding framework region residues from a non-humanimmunoglobulin that has the desired specificity, affinity, and/orcapability. In some embodiments, the humanized antibody is furthermodified by the substitution of additional residues either in the Fvframework region and/or within the replaced non-human residues to refineand optimize antibody specificity, affinity, and/or capability. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two or three, variable domains containing all,or substantially all, of the CDRs that correspond to the non-humanimmunoglobulin whereas all, or substantially all, of the frameworkregions are those of a human immunoglobulin consensus sequence. In someembodiments, the humanized antibody can also comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. In certain embodiments, such humanized antibodiesare used therapeutically because they should be less antigenic and mayreduce HAMA (human anti-mouse antibody) responses when administered to ahuman subject. One skilled in the art would be able to obtain afunctional humanized antibody with reduced immunogenicity followingknown techniques (see, e.g., U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; and 5,693,762).

In some embodiments, the invention provides an antibody thatspecifically binds the extracellular domain of human DLL4, wherein theantibody comprises one, two, three, four, five, and/or six of the CDRsof antibodies 21M18, 21M18 H9L2, and/or 21M18 H7L2. These antibodieshave been described in U.S. Pat. No. 7,750,124, filed Sep. 28, 2007.Antibodies 21M18 H7L2 and 21M18 H9L2 are humanized versions of themurine 21M18 antibody. Antibody 21M18 H7L2 is also referred to asOMP-21M18 and demcizumab.

In certain embodiments, the invention provides a DLL4 antagonist,wherein the antagonist is a DLL4 antibody that specifically binds anepitope within amino acids 27-217 of the extracellular domain of humanDLL4, and wherein the antibody comprises: a heavy chain CDR1 comprisingTAYYIH (SEQ ID NO: 1), a heavy chain CDR2 comprising YISCYNGATNYNQKFKG(SEQ ID NO:2), YISSYNGATNYNQKFKG (SEQ ID NO:3), or YISVYNGATNYNQKFKG(SEQ ID NO:4), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ IDNO:5). In some embodiments, the antibody further comprises a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:8). In some embodiments, the antibody comprisesa light chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:8). In some embodiments, the DLL4antibody comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO: 1),a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and aheavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:5); and a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2comprising AASNQGS (SEQ ID NO:7), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:8).

In certain embodiments, the invention provides an antibody thatspecifically binds an epitope within amino acids 27-217 of theextracellular domain of human DLL4, wherein the antibody comprises aheavy chain variable region having at least about 80% sequence identityto SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11, and/or a light chainvariable region having at least 80% sequence identity to SEQ ID NO: 12.In certain embodiments, the antibody comprises a heavy chain variableregion having at least about 85%, at least about 90%, at least about95%, at least about 97%, or at least about 99% sequence identity to SEQID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. In certain embodiments, theantibody comprises a light chain variable region having at least about85%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% sequence identity to SEQ ID NO: 12. In certainembodiments, the antibody comprises a heavy chain variable region havingat least about 95% sequence identity to SEQ ID NO:9, and/or a lightchain variable region having at least about 95% sequence identity to SEQID NO: 12. In certain embodiments, the antibody comprises a heavy chainvariable region comprising SEQ ID NO:9, and/or a light chain variableregion comprising SEQ ID NO: 12. In certain embodiments, the antibodycomprises a heavy chain variable region comprising SEQ ID NO:9 and alight chain variable region comprising SEQ ID NO: 12. In certainembodiments, the antibody comprises a heavy chain variable region havingat least about 95% sequence identity to SEQ ID NO: 10, and/or a lightchain variable region having at least about 95% sequence identity to SEQID NO: 12. In certain embodiments, the antibody comprises a heavy chainvariable region comprising SEQ ID NO: 10, and/or a light chain variableregion comprising SEQ ID NO: 12. In certain embodiments, the antibodycomprises a heavy chain variable region comprising SEQ ID NO: 10 and alight chain variable region comprising SEQ ID NO: 12. In certainembodiments, the antibody comprises a heavy chain variable region havingat least about 95% sequence identity to SEQ ID NO: 11, and/or a lightchain variable region having at least about 95% sequence identity to SEQID NO: 12. In certain embodiments, the antibody comprises a heavy chainvariable region comprising SEQ ID NO: 11, and/or a light chain variableregion comprising SEQ ID NO: 12. In certain embodiments, the antibodycomprises a heavy chain variable region comprising SEQ ID NO: 11 and alight chain variable region comprising SEQ ID NO: 12.

In certain embodiments, the anti-DLL4 antibody is the antibody producedby the hybridoma deposited with ATCC on Sep. 28, 2007 and having ATCCdeposit number PTA-8670, also known as murine 21M18. The murine 21M18antibody is described in detail in U.S. Pat. No. 7,750,124, filed Sep.28, 2007.

In certain embodiments, the anti-DLL4 antibody is the antibody encodedby the plasmid deposited with ATCC on May 10, 2007, having ATCC depositnumber PTA-8425, also known as 21M18 H7L2 and OMP-21M18. The OMP-21M18antibody is described in detail in U.S. Pat. No. 7,750,124, filed Sep.28, 2007. The anti-DLL4 antibody OMP-21M18 comprises a heavy chainvariable region comprising CDR amino acid sequences CDR1 (SEQ ID NO: 1);CDR2 (SEQ ID NO:3); and CDR3 (SEQ ID NO:5); and a light chain variableregion comprising CDR amino acid sequences CDR1 (SEQ ID NO:6); CDR2 (SEQID NO:7); and CDR3 (SEQ ID NO:8). In one embodiment, the OMP-21M18antibody comprises the heavy chain variable region sequence of SEQ IDNO: 10 and the light chain variable region sequence of SEQ ID NO: 12.

In certain embodiments, the anti-DLL4 antibody is the antibody encodedby the plasmid deposited with ATCC on May 10, 2007, having ATCC depositnumber PTA-8427, also known as 21M18 H9L2. The 21M18 H9L2 antibody isdescribed in detail in U.S. Pat. No. 7,750,124, filed Sep. 28, 2007.

In certain embodiments, the DLL4 antagonist (e.g., an antibody) binds tothe same epitope that an antibody comprising the heavy chain variableregion comprising SEQ ID NO: 10, and/or a light chain variable regioncomprising SEQ ID NO: 12 binds. In certain embodiments, the DLL4antagonist (e.g., an antibody) binds to the same epitope that anantibody comprising the heavy chain variable region comprising SEQ IDNO:9, and/or a light chain variable region comprising SEQ ID NO: 12binds. In certain embodiments, the DLL4 antagonist (e.g., an antibody)binds to the same epitope that an antibody comprising the heavy chainvariable region comprising SEQ ID NO: 11, and/or a light chain variableregion comprising SEQ ID NO: 12 binds. In some embodiments, the DLL4antagonist or antibody binds to the same epitope as murine antibody21M18. In some embodiments, the DLL4 antagonist or antibody binds to thesame epitope as humanized antibody 21M18 H7L2 (OMP-21M18). In someembodiments, the DLL4 antagonist or antibody binds to the same epitopeas humanized antibody 21M18 H9L2.

In certain embodiments, the DLL4 antagonist (e.g., an antibody) competesfor specific binding to an extracellular domain of human DLL4 with anantibody, wherein the antibody comprises a heavy chain variable regioncomprising SEQ ID NO: 10, and/or a light chain variable regioncomprising SEQ ID NO: 12. In certain embodiments, the DLL4 antagonist(e.g., an antibody) competes for specific binding to an extracellulardomain of human DLL4 with an antibody, wherein the antibody comprises aheavy chain variable region comprising SEQ ID NO:9, and/or a light chainvariable region comprising SEQ ID NO: 12. In certain embodiments, theDLL4 antagonist (e.g., an antibody) competes for specific binding to anextracellular domain of human DLL4 with an antibody, wherein theantibody comprises a heavy chain variable region comprising SEQ ID NO:11, and/or a light chain variable region comprising SEQ ID NO: 12. Insome embodiments, the DLL4 antagonist competes for specific binding toan extracellular domain of human DLL4 with an antibody encoded by theplasmid deposited with ATCC having deposit no. PTA-8425. In someembodiments, the DLL4 antagonist or antibody competes for specificbinding to an extracellular domain of human DLL4 with an antibodyencoded by the plasmid deposited with ATCC having deposit no. PTA-8427.In some embodiments, the DLL4 antagonist or antibody competes forspecific binding to an extracellular domain of human DLL4 with anantibody produced by the hybridoma deposited with ATCC having depositno. PTA-8670. In some embodiments, the DLL4 antagonist or antibodycompetes for specific binding to an epitope within amino acids 27-217 ofthe extracellular domain of human DLL4 in a competitive binding assay.Other anti-DLL4 antibodies are known in the art and may be used in themethods described herein.

In certain embodiments, the DLL4 antagonist is a human antibody. Humanantibodies can be directly prepared using various techniques known inthe art. In some embodiments, human antibodies may be generated fromimmortalized human B lymphocytes immunized in vitro or from lymphocytesisolated from an immunized individual. In either case, cells thatproduce an antibody directed against a target antigen can be generatedand isolated (see, e.g., Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77; Boemer et al., 1991, J. Immunol.,147:86-95; and U.S. Pat. Nos. 5,750,373; 5,567,610; and 5,229,275).

In some embodiments, the human antibody can be selected from a phagelibrary, wherein the phage library expresses human antibodies (Vaughanet al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998,PNAS, 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381;Marks et al., 1991, J. Mol. Biol., 222:581). Alternatively, phagedisplay technology can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. Techniques for the generation and use ofantibody phage libraries are described in U.S. Pat. Nos. 5,969,108;6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2008, J. Mol. Bio., 376:1182-1200.

Once antibodies are identified, affinity maturation strategies known inthe art, including but not limited to, chain shuffling (Marks et al.,1992, Bio/Technology, 10:779-783) and site-directed mutagenesis, may beemployed to generate high affinity human antibodies.

In some embodiments, human antibodies can be made in transgenic micethat contain human immunoglobulin loci. Upon immunization these mice arecapable of producing the full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. This approach isdescribed in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016.

In certain embodiments, the DLL4 antagonist is a bispecific antibody.Bispecific antibodies are capable of specifically recognizing andbinding to at least two different epitopes. The different epitopes caneither be within the same molecule or on different molecules. In someembodiments, the antibodies can specifically recognize and bind a firstantigen target, (e.g., DLL4) as well as a second antigen target, such asan effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or B7) or aFc receptor (e.g., CD64, CD32, or CD16) so as to focus cellular defensemechanisms to the cell expressing the first antigen target. In someembodiments, the antibodies can be used to direct cytotoxic agents tocells which express a particular target antigen, such as DLL4. Theseantibodies possess an antigen-binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,or TETA. In certain embodiments, the antibodies can be used to affectangiogenesis. In certain embodiments, the bispecific antibodyspecifically binds DLL4, as well as a second Notch ligand (e.g., Jagged1or Jagged2), or at least one Notch receptor selected from the groupconsisting of Notch1, Notch2, Notch3, and Notch4. In certainembodiments, the bispecific antibody specifically binds DLL4, as well asVEGF. In some embodiments, the bispecific antibody is a bispecificantibody disclosed in U.S. patent application Ser. No. 13/625,417, filedon Sep. 24, 2012. In some embodiments, the anti-VEGF/DLL4 bispecificantibody is 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, or219R45-MB-21R83 as disclosed in U.S. patent application Ser. No.13/625,417, filed on Sep. 24, 2012.

Techniques for making bispecific antibodies are known by those skilledin the art, see for example, Millstein et al., 1983, Nature,305:537-539; Brennan et al., 1985, Science, 229:81; Suresh et al, 1986,Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBO J.,10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225; Kostelnyet al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994, J.Immunol., 152:5368; U.S. Pat. No. 5,731,168; and U.S. Patent ApplicationPub. No. 2011/0123532). Bispecific antibodies can be intact antibodiesor antibody fragments. Antibodies with more than two valencies are alsocontemplated. For example, trispecific antibodies can be prepared (Tuttet al., 1991, J. Immunol., 147:60). Thus, in certain embodiments theantibodies to DLL4 are multispecific.

In certain embodiments, the DLL4 antagonists (e.g., antibodies or otherpolypeptides) described herein may be monospecific. For example, incertain embodiments, each of the one or more antigen-binding sites thatan antibody contains is capable of binding (or binds) a homologousepitope on DLL4.

In certain embodiments, the DLL4 antagonist is an antibody fragment.Antibody fragments may have different functions or capabilities thanintact antibodies; for example, antibody fragments can have increasedtumor penetration. Various techniques are known for the production ofantibody fragments including, but not limited to, proteolytic digestionof intact antibodies. In some embodiments, antibody fragments include aF(ab′)2 fragment produced by pepsin digestion of an antibody molecule.In some embodiments, antibody fragments include a Fab fragment generatedby reducing the disulfide bridges of an F(ab′)2 fragment. In otherembodiments, antibody fragments include a Fab fragment generated by thetreatment of the antibody molecule with papain and a reducing agent. Incertain embodiments, antibody fragments are produced recombinantly. Insome embodiments, antibody fragments include Fv or single chain Fv(scFv) fragments. Fab, Fv, and scFv antibody fragments can be expressedin, and secreted from, E. coli or other host cells, allowing for theproduction of large amounts of these fragments. In some embodiments,antibody fragments are isolated from antibody phage libraries asdiscussed herein. For example, methods can be used for the constructionof Fab expression libraries (Huse et al., 1989, Science, 246:1275-1281)to allow rapid and effective identification of monoclonal Fab fragmentswith the desired specificity for DLL4, or derivatives, fragments,analogs or homologs thereof. In some embodiments, antibody fragments arelinear antibody fragments. In certain embodiments, antibody fragmentsare monospecific or bispecific. In certain embodiments, the DLL4antagonist is a scFv. Various techniques can be used for the productionof single-chain antibodies specific to DLL4 (see, e.g., U.S. Pat. No.4,946,778).

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

For the purposes of the present invention, it should be appreciated thatmodified antibodies, or fragments thereof, can comprise any type ofvariable region that provides for the association of the antibody withDLL4. In this regard, the variable region may be derived from any typeof mammal that can be induced to mount a humoral response and generateimmunoglobulins against a desired antigen (e.g., DLL4). As such, thevariable region of the modified antibodies can be, for example, ofhuman, murine, non-human primate (e.g., cynomolgus monkeys, macaques,etc.) or lepine origin. In some embodiments, both the variable andconstant regions of the modified immunoglobulins are human. In otherembodiments, the variable regions of compatible antibodies (usuallyderived from a non-human source) can be engineered or specificallytailored to improve the binding properties or reduce the immunogenicityof the molecule. In this respect, variable regions useful in the presentinvention can be humanized or otherwise altered through the inclusion ofimported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencemodification. Although the CDRs may be derived from an antibody of thesame class or even subclass as the antibody from which the frameworkregions are derived, it is envisaged that the CDRs will be derived froman antibody of a different class and preferably from an antibody from adifferent species. It may not be necessary to replace all of the CDRswith all of the CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are necessary tomaintain the activity of the antigen binding site.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or antigen-bindingfragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics, such as increased tumorlocalization, increased tumor penetration, reduced serum half-life orincreased serum half-life when compared with an antibody ofapproximately the same immunogenicity comprising a native or unalteredconstant region. In some embodiments, the constant region of themodified antibodies comprises a human constant region. Modifications tothe constant region include additions, deletions, or substitutions ofone or more amino acids in one or more domains. The modified antibodiesdisclosed herein may comprise alterations or modifications to one ormore of the three heavy chain constant domains (CH1, CH2 or CH3) and/orto the light chain constant domain (CL). In some embodiments, one ormore domains are partially or entirely deleted from the constant regionsof the modified antibodies. In some embodiments, the entire CH2 domainhas been removed (ACH2 constructs). In some embodiments, the omittedconstant region domain is replaced by a short amino acid spacer (e.g.,10 aa residues) that provides some of the molecular flexibilitytypically imparted by the absent constant region.

In certain embodiments, the modified antibodies are engineered to fusethe CH3 domain directly to the hinge region of the antibody. In otherembodiments, a peptide spacer is inserted between the hinge region andthe modified CH2 and/or CH3 domains. For example, constructs may beexpressed wherein the CH2 domain has been deleted and the remaining CH3domain (modified or unmodified) is joined to the hinge region with a5-20 amino acid spacer. Such a spacer may be added to ensure that theregulatory elements of the constant domain remain free and accessible orthat the hinge region remains flexible. However, it should be noted thatamino acid spacers can, in some cases, prove to be immunogenic andelicit an unwanted immune response against the construct. Accordingly,in certain embodiments, any spacer added to the construct will berelatively non-immunogenic so as to maintain the desired biologicalqualities of the modified antibodies.

In some embodiments, the modified antibodies may have only a partialdeletion of a constant domain or substitution of a few or even a singleamino acid. For example, the mutation of a single amino acid in selectedareas of the CH2 domain may be enough to substantially reduce Fc bindingand thereby increase tumor localization and/or tumor penetration.Similarly, it may be desirable to simply delete the part of one or moreconstant region domains that control a specific effector function (e.g.,complement C1q binding) to be modulated. Such partial deletions of theconstant regions may improve selected characteristics of the antibody(serum half-life) while leaving other desirable functions associatedwith the subject constant region domain intact. Moreover, as alluded toabove, the constant regions of the disclosed antibodies may be modifiedthrough the mutation or substitution of one or more amino acids thatenhances the profile of the resulting construct. In this respect it maybe possible to disrupt the activity provided by a conserved binding site(e.g., Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. In certain embodiments,the modified antibodies comprise the addition of one or more amino acidsto the constant region to enhance desirable characteristics such asdecreasing or increasing effector function or provide for more cytotoxinor carbohydrate attachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the Cl component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan antibody can bind to a cell expressing a Fc receptor (FcR). There area number of Fc receptors which are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells, release of inflammatorymediators, placental transfer and control of immunoglobulin production.

In certain embodiments, the DLL4 antibodies provide for altered effectorfunctions that, in turn, affect the biological profile of theadministered antibody. For example, in some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating modifiedantibody (e.g., DLL4 antibody) thereby increasing tumor localizationand/or penetration. In other embodiments, the constant regionmodifications increase or reduce the serum half-life of the antibody. Insome embodiments, the constant region is modified to eliminate disulfidelinkages or oligosaccharide moieties allowing for enhanced tumorlocalization and/or penetration.

In certain embodiments, a DLL4 antibody does not have one or moreeffector functions. In some embodiments, the antibody has noantibody-dependent cellular cytoxicity (ADCC) activity and/or nocomplement-dependent cytoxicity (CDC) activity. In certain embodiments,the antibody does not bind to an Fc receptor and/or complement factors.In certain embodiments, the antibody has no effector function.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized, and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids.

Thus, the present invention provides methods for generating an antibodythat binds the extracellular domain of human DLL4. In some embodiments,the method for generating an antibody that binds DLL4 comprises usinghybridoma techniques. In some embodiments, the method comprises using anextracellular domain of mouse DLL4 or human DLL4 as an immunizingantigen. In some embodiments, the method of generating an antibody thatbinds DLL4 comprises screening a human phage library. The presentinvention further provides methods of identifying an antibody that bindsto DLL4. In some embodiments, the antibody is identified by screeningfor binding to DLL4 with flow cytometry (FACS). In some embodiments, theantibody is screened for binding to human DLL4. In some embodiments, theantibody is screened for binding to mouse DLL4. In some embodiments, theantibody is identified by screening for inhibition or blocking ofDLL4-induced Notch activation. In some embodiments, the DLL4 is humanDLL4. In some embodiments, the Notch is human Notch1, Notch2, Notch3, orNotch4.

In certain embodiments, the antibodies described herein are isolated. Incertain embodiments, the antibodies described herein are substantiallypure.

Certain anti-DLL4 antibodies have been described, for example, in U.S.Pat. No. 7,750,124. Additional anti-DLL4 antibodies are described in,e.g., International Patent Publication Nos. WO 2008/091222 and WO2008/0793326, and U.S. Patent Application Publication Nos. 2008/0014196,2008/0175847, 2008/0181899, 2008/0107648, and 2010/0196385.

In some embodiments of the present invention, the DLL4 antagonists arepolypeptides. The polypeptides can be recombinant polypeptides, naturalpolypeptides, or synthetic polypeptides that bind an epitope comprisingamino acids within the extracellular domain of human DLL4. In someembodiments, the polypeptides comprise an antibody or fragment thereofthat binds an epitope within the extracellular domain of human DLL4. Itwill be recognized by those of skill in the art that some amino acidsequences of a polypeptide can be varied without significant effect onthe structure or function of the protein. Thus, the polypeptides furtherinclude variations of the polypeptides which show substantial bindingactivity to an epitope of the human DLL4 protein. In some embodiments,amino acid sequence variations of polypeptides include deletions,insertions, inversions, repeats, and/or type substitutions.

The polypeptides and variants thereof, can be further modified tocontain additional chemical moieties not normally part of thepolypeptide. The derivatized moieties can improve the solubility, thebiological half-life, or the absorption of the polypeptide. The moietiescan also reduce or eliminate any undesirable side effects of thepolypeptides and variants. An overview for such chemical moieties can befound in Remington: The Science and Practice of Pharmacy, 21st Edition,2005, University of the Sciences in Philadelphia, Pa.

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthesis methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitable host.In some embodiments, a DNA sequence is constructed using recombinanttechnology by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional variantsthereof.

In some embodiments, a DNA sequence encoding a polypeptide of interestmay be constructed by chemical synthesis using an oligonucleotidesynthesizer. Oligonucleotides can be designed based on the amino acidsequence of the desired polypeptide and by selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence encoding a polypeptide of interest. Forexample, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular polypeptide can be synthesized. Forexample, several small oligonucleotides coding for portions of thedesired polypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ and/or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the polynucleotide sequences encoding a particular polypeptideof interest can be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe polypeptide in a desired host. Proper assembly can be confirmed bynucleotide sequencing, restriction mapping, and/or expression of abiologically active polypeptide in a suitable host. As is well-known inthe art, in order to obtain high expression levels of a transfected genein a host, the gene must be operatively linked to transcriptional andtranslational expression control sequences that are functional in thechosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding DLL4 antagonists such as polypeptidesor antibodies, or fragments thereof. For example, recombinant expressionvectors can be replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a polypeptide chain of an anti-DLL4antibody, or fragment thereof, operatively linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral, or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a regulatory element or elementshaving a role in gene expression, for example, transcriptional promotersand/or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences.Regulatory elements can include an operator sequence to controltranscription. The ability to replicate in a host, usually conferred byan origin of replication, and a selection gene to facilitate recognitionof transformants can also be incorporated. DNA regions are “operativelylinked” when they are functionally related to each other. For example,DNA for a signal peptide (secretory leader) is operatively linked to DNAfor a polypeptide if it is expressed as a precursor which participatesin the secretion of the polypeptide; a promoter is operatively linked toa coding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of an expression vector and control elements depends upon thechoice of host. A wide variety of expression host/vector combinationscan be employed. Useful expression vectors for eukaryotic hosts include,for example, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Usefulexpression vectors for bacterial hosts include known bacterial plasmids,such as plasmids from E. coli, including pCR1, pBR322, pMB9 and theirderivatives, and wider host range plasmids, such as M13 and otherfilamentous single-stranded DNA phages.

Suitable host cells for expression of a DLL4 antagonist polypeptide orantibody (or a DLL4 protein to use as an antigen) include prokaryotes,yeast, insect, or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram-negative orgram-positive organisms, for example, E. coli or Bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems can also be employed.

Various mammalian or insect cell culture systems are used to expressrecombinant protein. Expression of recombinant proteins in mammaliancells may be preferred because such proteins are generally correctlyfolded, appropriately modified, and biologically functional. Examples ofsuitable mammalian host cell lines include COS-7 (monkeykidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammarytumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamsterovary-derived), HeLa (human cervical cancer-derived), BHK (hamsterkidney fibroblast-derived) cell lines, and HEK-293 (human embryonickidney-derived) cell lines and variants thereof. Mammalian expressionvectors can comprise non-transcribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′or 3′ non-translated sequences, such as necessary ribosome bindingsites, a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are well-known tothose of skill in the art (see, e.g., Luckow and Summers, 1988,Bio/Technology, 6:47).

The proteins produced by a transformed host can be purified according toany suitable method. Such methods include chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Affinity tags such as hexa-histidine, maltose bindingdomain, influenza coat sequence and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Isolated proteins can be physicallycharacterized using such techniques as proteolysis, high performanceliquid chromatography (HPLC), nuclear magnetic resonance (NMR), andx-ray crystallography.

For example, supernatants from expression systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin isemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step is employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite media is employed, including but not limited to, ceramichydroxyapatite (CHT). In some embodiments, one or more reversed-phaseHPLC steps employing hydrophobic RP-HPLC media, (e.g., silica gel havingpendant methyl or other aliphatic groups), is employed to further purifya protein. Some or all of the foregoing purification steps, in variouscombinations, can be employed to provide a homogeneous recombinantprotein.

In some embodiments, recombinant protein produced in bacterial cultureis isolated, for example, by initial extraction from cell pellets,followed by one or more concentration, salting-out, aqueous ionexchange, or size exclusion chromatography steps. In certainembodiments, HPLC is employed for final purification steps. Microbialcells employed in expression of a recombinant protein can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent ApplicationPub. Nos. 2008/0312425, 2009/0187005, and U.S. Pat. No. 7,691,980.

In certain embodiments, the DLL4 antagonist is a polypeptide that is notan antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, 2007, Curr. Opin.Biotechnol., 18:295-304; Hosse et al., 2006, Protein Science, 15:14-27;Gill et al., 2006, Curr. Opin. Biotechnol., 17:653-658; Nygren, 2008,FEBS J., 275:2668-76; and Skerra, 2008, FEBS J., 275:2677-83. In certainembodiments, phage display technology may be used to produce and/oridentify a DLL4 antagonist polypeptide. In certain embodiments, the DLL4antagonist polypeptide comprises a protein scaffold of a type selectedfrom the group consisting of protein A, protein G, a lipocalin, afibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

In certain embodiments, the DLL4 antagonists or antibodies can be usedin any one of a number of conjugated (e.g., an immunoconjugate orradioconjugate) or non-conjugated forms. In certain embodiments, theantibodies are used in non-conjugated form to harness the subject'snatural defense mechanisms including CDC and/or ADCC to eliminatemalignant or cancerous cells.

In certain embodiments, the DLL4 antagonist (e.g., an antibody orpolypeptide) is conjugated to a cytotoxic agent. In some embodiments,the cytotoxic agent is a chemotherapeutic agent including, but notlimited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents. In someembodiments, the cytotoxic agent is a enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof,including but not limited to, diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, restrictocin, phenomycin, enomycin, andthe tricothecenes. In certain embodiments, the cytotoxic agent is aradioactive isotope to produce a radioconjugate or a radioconjugatedantibody. A variety of radionuclides are available for the production ofradioconjugated antibodies including, but not limited to, ⁹⁰Y, ¹²⁵I,¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re and ²¹²Bi. Conjugates of an antibody and one or more smallmolecule toxins, such as a calicheamicin, maytansine, maytansinoids, atrichothene, and CC1065, and the derivatives of these toxins that havetoxin activity, can also be used. Conjugates of an antibody andcytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents.

In some embodiments, the DLL4 antagonist is a non-protein molecule. Incertain embodiments, the DLL4 antagonist is a small molecule.

In vivo and in vitro assays for determining whether an agent is a DLL4antagonist are known in the art. In some embodiments, a cell-based,luciferase reporter assay utilizing a TCF/Luc reporter vector containingmultiple copies of the TCF-binding domain upstream of a fireflyluciferase reporter gene may be used to measure DLL4-induced Notchsignaling levels in vitro. In other embodiments, a cell-based,luciferase reporter assay utilizing a CBF/Luc reporter vector containingmultiple copies of the CBF-binding domain upstream of a fireflyluciferase reporter gene may be used. The level of Notch activationinduced by DLL4 in the presence of a DLL4 antagonist is compared to thelevel of Notch activation induced by DLL4 in the absence of the DLL4antagonist. In some embodiments, the effect of a DLL4 antagonist onDLL4/Notch signaling can be assessed by measuring the effect of theagent on the expression level of one or more Notch pathway target genes.

III. Methods of Use and Pharmaceutical Compositions

The present invention provides methods of treating diseases such ascancer with a DLL4 antagonist, while screening for, monitoring,preventing, and/or controlling side effects and/or toxicities,including, but not limited to cardiovascular side effects and/ortoxicities associated with the DLL4 antagonist. Side effects and/ortoxicities associated with cancer treatment may include, but are notlimited to, fatigue, vomiting, nausea, diarrhea, pain, hair loss,neutropenia, anemia, thrombocytopenia, cardiovascular complications, andany combination thereof. Cardiovascular complications (e.g.,cardiovascular side effects and/or toxicities) may be grouped into threemain categories: 1) vascular conditions, 2) cardiac structural problems,and 3) cardiac dysfunction and heart failure. As used herein, “vascularconditions” include but are not limited to, atherosclerosis,hypertension, arterial thrombosis, vasculitis, and deep venousthrombosis/pulmonary embolus. As used herein, “cardiac structuralproblems” and “cardiac dysfunction and heart failure” include but arenot limited to, valvular heart disease, pericardial effusion,pericardial constriction, angina, coronary artery disease,cardiomyopathy, myocardial ischemia, myocardial infarction (MI),arrhythmias, myocarditis, left ventricular dysfunction, heart failure,congestive heart failure (CHF), and combinations thereof. Cardiacstructural problems, cardiac dysfunction, and heart failure directlyimpact the heart, while, generally, vascular conditions (e.g.,hypertension) are a more systemic complication. As used herein,“cardiotoxicity” refers to cardiac structural problems, cardiacdysfunction, and heart failure. Thus, in some aspects and/or embodimentsof the methods described herein, the screening for, monitoring,preventing, and/or controlling cardiovascular side effects and/ortoxicities is screening for, monitoring, preventing, and/or controllingcardiotoxicity. Often cardiotoxicity is asymptomatic and/or early signsof cardiotoxicity are not evident with, for example, Dopplerechocardiograms and/or with left ventricular ejection fraction (LVEF)monitoring.

Natriuretic peptides are produced by the heart and vasculature andinclude 4 identified types. A-type natriuretic peptide (ANP) is secretedlargely by the atrial myocardium, B-type natriuretic peptide (BNP) isproduced mainly by the ventricular myocardium, C-type natriureticpeptide (CNP) is produced by endothelial cells that line the bloodvessels, and D-type natriuretic peptide has been isolated in plasma andatrial myocardium. The precursor prohormone of each natriuretic peptideis encoded by a separate gene. proBNP is a 108 amino acid peptide thatis cleaved by the proteolytic enzyme furin into a 32 amino acidC-terminal peptide (BNP) and a 76 amino acid N-terminal peptide(NT-proBNP). proANP is a 126 amino acid peptide that is cleaved by theserine protease enzyme corin into a 28 amino acid C-terminal peptide(ANP) and a 98 amino acid N-terminal peptide (NT-proANP). Increases inthe levels of natriuretic peptides have been used in the diagnosis ofheart failure. Accordingly, in some embodiments, the present inventionprovides methods for using natriuretic peptide levels to monitorcardiotoxicity in subjects being treated with a DLL4 antagonist. In someembodiments, the methods use natriuretic peptide levels to monitorand/or detect acute cardiotoxicity. In some embodiments, monitoring thelevel of a natriuretic peptide gives an early indication ofcardiotoxicity and/or congestive heart failure (CHF). In someembodiments, the methods detect cardiotoxicity prior to any evidence ofcardiac dysfunction as evaluated by Doppler echocardiograms and/or withLVEF monitoring.

In certain embodiments, the cardiovascular side effects and/ortoxicities that are detected, identified, monitored, reduced, prevented,attenuated, and/or screened for are cardiovascular side effects and/ortoxicities caused by, associated with, and/or related to administrationof a DLL4 antagonist or treatment with a DLL4 antagonist. In certainembodiments, the cardiovascular side effects and/or toxicities arerelated to the DLL4 antagonist.

In certain embodiments, the cardiovascular side effects and/ortoxicities (e.g., side effect and/or toxicity related to treatment witha DLL4 antagonist) that is detected, identified, monitored, reduced,prevented, attenuated, and/or screened for in a method described hereindoes not include hypertension. In some embodiments the cardiovascularside effect and/or toxicity does not include a vascular condition. Incertain embodiments, the cardiovascular side effect and/or toxicity thatis detected, identified, monitored, reduced, prevented, attenuated,and/or screened for in a method described herein is a cardiotoxicitysuch as a cardiac structural problem, cardiac dysfunction, or heartfailure. In certain embodiments, the cardiotoxicity is left ventriculardysfunction. In certain embodiments, the cardiotoxicity is congestiveheart failure. In some embodiments, the cardiotoxicity is in an early orreversible stage of development.

The invention provides methods for selecting a subject for treatmentwith a DLL4 antagonist, comprising: determining the level of a biomarkerin a sample, and selecting the subject for treatment with the DLL4antagonist if the level of the biomarker is below a predetermined level.In some embodiments, the methods for selecting a subject for treatmentwith a DLL4 antagonist, comprise: obtaining a biological sample from thesubject, determining the level of a biomarker in the sample, andselecting the subject for treatment with the DLL4 antagonist if thelevel of the biomarker is below a predetermined level. In someembodiments, the biomarker is a natriuretic peptide. In someembodiments, the natriuretic peptide is an atrial natriuretic peptide(ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, orvariants thereof. ANP-type peptides include pre-proANP, proANP,NT-proANP, and ANP. BNP-type peptides include pre-proBNP, proBNP,NT-proBNP, and BNP. In some embodiments, the natriuretic peptide is BNP.In some embodiments, the natriuretic peptide is NT-proBNP. In someembodiments, the natriuretic peptide is ANP.

In some embodiments, the method of selecting a subject for treatmentwith a DLL4 antagonist comprises: obtaining a biological sample from thesubject, determining the level of a natriuretic peptide in the sample,and selecting the subject for treatment with the DLL4 antagonist if thelevel of the natriuretic peptide is below a predetermined level. In someembodiments, the biological sample is blood, serum, or plasma. In someembodiments, the natriuretic peptide is B-type natriuretic peptide(BNP). Thus, in some embodiments, the methods of selecting a subject fortreatment with a DLL4 antagonist, comprising: obtaining a biologicalsample from the subject, determining the level of BNP in the sample, andselecting the subject for treatment with the DLL4 antagonist if thelevel of BNP is below a predetermined level.

The invention also provides methods of identifying a subject as eligiblefor treatment with a DLL4 antagonist, comprising: determining the levelof a biomarker in a sample, and identifying the subject as eligible fortreatment with the DLL4 antagonist if the level of the biomarker isbelow a predetermined level. In some embodiments, the methods ofidentifying a subject as eligible for treatment with a DLL4 antagonistcomprise: obtaining a biological sample from the subject, determiningthe level of a biomarker in the sample, and identifying the subject aseligible for treatment with the DLL4 antagonist if the level of thebiomarker is below a predetermined level. In some embodiments, thebiomarker is a natriuretic peptide. In some embodiments, the natriureticpeptide is an atrial natriuretic peptide (ANP)-type peptides, a brainnatriuretic peptide (BNP)-type peptides, or variants thereof. ANP-typepeptides include pre-proANP, proANP, NT-proANP, and ANP. BNP-typepeptides include pre-proBNP, proBNP, NT-proBNP, and BNP. In someembodiments, the natriuretic peptide is BNP. In some embodiments, thenatriuretic peptide is NT-proBNP. In some embodiments, the methods ofidentifying a subject as eligible for treatment with a DLL4 antagonistcomprise: obtaining a biological sample from the subject, determiningthe level of BNP in the sample, and identifying the subject as eligiblefor treatment with the DLL4 antagonist if the level of BNP is below apredetermined level.

The invention also provides methods of monitoring a subject receivingtreatment with a DLL4 antagonist for the development of cardiovascularside effects and/or toxicity, comprising: determining the level of abiomarker in a sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein an increase inthe level of the biomarker indicates development of cardiovascular sideeffects and/or toxicity. In some embodiments, the methods of monitoringa subject receiving treatment with a DLL4 antagonist for the developmentof cardiovascular side effects and/or toxicity comprise: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of thebiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof cardiovascular side effects and/or toxicity. In some embodiments, thecardiovascular side effect and/or toxicity is cardiotoxicity. In someembodiments, the biomarker is a natriuretic peptide. In someembodiments, the natriuretic peptide is an atrial natriuretic peptide(ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, orvariants thereof. ANP-type peptides include pre-proANP, proANP,NT-proANP, and ANP. BNP-type peptides include pre-proBNP, proBNP,NT-proBNP, and BNP. In some embodiments, the natriuretic peptide is BNP.In some embodiments, the natriuretic peptide is NT-proBNP. In someembodiments, the natriuretic peptide is ANP. In some embodiments, amethod of monitoring a subject receiving treatment with a DLL4antagonist for the development of cardiotoxicity, comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of BNP in the sample, and comparing the level of BNP in the sampleto a predetermined level of BNP, wherein an increase in the level of BNPindicates development of cardiotoxicity.

The invention also provides methods of detecting the development ofcardiovascular side effects and/or toxicity in a subject receivingtreatment with a DLL4 antagonist, comprising: determining the level of abiomarker in a sample, and comparing the level of a biomarker in thesample to a predetermined level of the biomarker, wherein an increase inthe level of the biomarker indicates development of cardiovascular sideeffects and/or toxicity. In some embodiments, the methods of detectingthe development of cardiovascular side effects and/or toxicity in asubject receiving treatment with a DLL4 antagonist comprise: obtaining abiological sample from the subject receiving treatment, determining thelevel of a biomarker in the sample, and comparing the level of abiomarker in the sample to a predetermined level of the biomarker,wherein an increase in the level of the biomarker indicates developmentof cardiovascular side effects and/or toxicity. In some embodiments, thecardiovascular side effect and/or toxicity is cardiotoxicity. In someembodiments, the biomarker is a natriuretic peptide. In someembodiments, the natriuretic peptide is an atrial natriuretic peptide(ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, orvariants thereof. ANP-type peptides include pre-proANP, proANP,NT-proANP, and ANP. BNP-type peptides include pre-proBNP, proBNP,NT-proBNP, and BNP. In some embodiments, the natriuretic peptide is BNP.In some embodiments, the natriuretic peptide is NT-proBNP. In someembodiments, the natriuretic peptide is ANP. In some embodiments, themethods of detecting the development of cardiotoxicity in a subjectreceiving treatment with a DLL4 antagonist comprise: obtaining abiological sample from the subject receiving treatment, determining thelevel of BNP in the sample, and comparing the level of BNP in the sampleto a predetermined level of BNP, wherein an increase in the level of BNPindicates development of cardiotoxicity.

The invention also provides methods for identifying cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist, comprising: determining the level of a biomarker in asample, and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then a cardiovascular side effect and/or toxicity isindicated. In some embodiments, the methods for identifyingcardiovascular side effects and/or toxicity in a subject receivingtreatment with a DLL4 antagonist comprise: obtaining a biological samplefrom the subject receiving treatment, determining the level of abiomarker in the sample, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then a cardiovascular side effect and/or toxicity isindicated. In some embodiments, the cardiovascular side effect and/ortoxicity is cardiotoxicity. In some embodiments, the biomarker is anatriuretic peptide. In some embodiments, the natriuretic peptide is anatrial natriuretic peptide (ANP)-type peptides, a brain natriureticpeptide (BNP)-type peptides, or variants thereof. ANP-type peptidesinclude pre-proANP, proANP, NT-proANP, and ANP. BNP-type peptidesinclude pre-proBNP, proBNP, NT-proBNP, and BNP. In some embodiments, thenatriuretic peptide is BNP. In some embodiments, the natriuretic peptideis NT-proBNP. In some embodiments, the natriuretic peptide is ANP. Insome embodiments, a method for identifying cardiotoxicity in a subjectreceiving treatment with a DLL4 antagonist comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of BNP in the sample, and comparing the level of BNP in the sampleto a predetermined level of BNP, wherein if the level of BNP in thesample is higher than the predetermined level of BNP then cardiotoxicityis indicated.

The invention also provides methods for monitoring cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist, comprising: determining the level of a biomarker in asample, and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then cardiovascular side effects and/or toxicity is indicated.In some embodiments, the methods for monitoring cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist comprise: obtaining a biological sample from the subjectreceiving treatment, determining the level of a biomarker in the sample,and comparing the level of the biomarker in the sample to apredetermined level of the biomarker, wherein if the level of thebiomarker in the sample is higher than the predetermined level of thebiomarker then cardiovascular side effects and/or toxicity is indicated.In some embodiments, the cardiovascular side effect and/or toxicity iscardiotoxicity. In some embodiments, the biomarker is a natriureticpeptide. In some embodiments, the natriuretic peptide is an atrialnatriuretic peptide (ANP)-type peptides, a brain natriuretic peptide(BNP)-type peptides, or variants thereof. ANP-type peptides includepre-proANP, proANP, NT-proANP, and ANP. BNP-type peptides includepre-proBNP, proBNP, NT-proBNP, and BNP. In some embodiments, thenatriuretic peptide is BNP. In some embodiments, the natriuretic peptideis NT-proBNP. In some embodiments, the natriuretic peptide is ANP. Insome embodiments, a method for monitoring cardiotoxicity in a subjectreceiving treatment with a DLL4 antagonist comprises: obtaining abiological sample from the subject receiving treatment, determining thelevel of BNP in the sample, and comparing the level of BNP in the sampleto a predetermined level of BNP, wherein if the level of BNP in thesample is higher than the predetermined level of BNP then cardiotoxicityis indicated.

The invention also provides methods of reducing cardiovascular sideeffects and/or toxicity in a subject receiving treatment with a DLL4antagonist, comprising: determining the level of a biomarker in a samplefrom the subject, comparing the level of the biomarker in the sample toa predetermined level of the biomarker, and administering to the subjecta therapeutically effective amount of a cardioprotective medication suchas an ACE inhibitor and/or a β-blocker if the level of the biomarker inthe sample is higher than the predetermined level of the biomarker. Insome embodiments, the methods of reducing cardiovascular side effectsand/or toxicity in a subject receiving treatment with a DLL4 antagonistcomprise: obtaining a biological sample from the subject receivingtreatment, determining the level of a biomarker in the sample, comparingthe level of the biomarker in the sample to a predetermined level of thebiomarker, and administering to the subject a therapeutically effectiveamount of a cardioprotective medication such as an ACE inhibitor and/ora β-blocker if the level of the biomarker in the sample is higher thanthe predetermined level of the biomarker. In some embodiments, thecardiovascular side effect and/or toxicity is cardiotoxicity. In someembodiments, the biomarker is a natriuretic peptide. In someembodiments, the natriuretic peptide is an atrial natriuretic peptide(ANP)-type peptides, a brain natriuretic peptide (BNP)-type peptides, orvariants thereof. ANP-type peptides include pre-proANP, proANP,NT-proANP, and ANP. BNP-type peptides include pre-proBNP, proBNP,NT-proBNP, and BNP. In some embodiments, the natriuretic peptide is BNP.In some embodiments, the natriuretic peptide is NT-proBNP. In someembodiments, the natriuretic peptide is ANP. In some embodiments, amethod for reducing cardiotoxicity in a subject receiving treatment witha DLL4 antagonist comprises: obtaining a biological sample from thesubject receiving treatment, determining the level of BNP in the sample,and comparing the level of BNP in the sample to a predetermined level ofBNP, and administering to the subject a therapeutically effective amountof a cardioprotective medication such as an ACE inhibitor and/or aβ-blocker if the level of BNP in the sample is higher than thepredetermined level of BNP.

The invention also provides methods of preventing or attenuating thedevelopment of cardiovascular side effects and/or toxicity in a subjectreceiving treatment with a DLL4 antagonist, comprising: determining thelevel of a biomarker in a sample from the subject, comparing the levelof the biomarker in the sample to a predetermined level of thebiomarker; administering to the subject a therapeutically effectiveamount of a cardioprotective medication such as an ACE inhibitor and/ora β-blocker, and administering to the subject the DLL4 antagonist. Insome embodiments, the methods of preventing or attenuating thedevelopment of cardiovascular side effects and/or toxicity in a subjectreceiving treatment with a DLL4 antagonist comprise: obtaining abiological sample from the subject prior to treatment with the DLL4antagonist, determining the level of a biomarker in the sample,comparing the level of the biomarker in the sample to a predeterminedlevel of the biomarker; administering to the subject a therapeuticallyeffective amount of a cardioprotective medication such as an ACEinhibitor and/or a β-blocker, and administering to the subject the DLL4antagonist. In some embodiments, the cardiovascular side effect and/ortoxicity is cardiotoxicity. In some embodiments, the biomarker is anatriuretic peptide. In some embodiments, the natriuretic peptide is anatrial natriuretic peptide (ANP)-type peptides, a brain natriureticpeptide (BNP)-type peptides, or variants thereof. ANP-type peptidesinclude pre-proANP, proANP, NT-proANP, and ANP. BNP-type peptidesinclude pre-proBNP, proBNP, NT-proBNP, and BNP. In some embodiments, thenatriuretic peptide is BNP. In some embodiments, the natriuretic peptideis NT-proBNP. In some embodiments, the natriuretic peptide is ANP. Insome embodiments, a method of preventing or attenuating the developmentof cardiotoxicity in a subject receiving treatment with a DLL4antagonist comprises: obtaining a biological sample from the subjectprior to treatment with the DLL4 antagonist, determining the level ofBNP in the sample, comparing the level of BNP in the sample to apredetermined level of BNP; administering to the subject atherapeutically effective amount of a cardioprotective medication suchas an ACE inhibitor and/or a β-blocker if the level of BNP in the sampleis higher than the predetermined level of BNP; and administering to thesubject the DLL4 antagonist.

In some embodiments of the methods described herein, the predeterminedlevel is about 300 pg/ml or less in a blood, serum, or plasma sample. Insome embodiments, the predetermined level is about 200 pg/ml or less ina blood, serum, or plasma sample. In some embodiments, the predeterminedlevel is about 250 pg/ml or less in a blood, serum, or plasma sample. Insome embodiments, the predetermined level is about 200 pg/ml or less ina blood, serum, or plasma sample. In some embodiments, the predeterminedlevel is about 150 pg/ml or less in a blood, serum, or plasma sample. Insome embodiments, the predetermined level is about 100 pg/ml or less ina blood, serum, or plasma sample. In the context of predetermined levelsof BNP, the term “about” means the referenced amount plus or minus 10%of that referenced amount.

In some embodiments, the predetermined level of a biomarker (e.g.,natriuretic peptide or BNP) is the amount of the biomarker in a sampleobtained at an earlier date. In some embodiments, the predeterminedlevel of a biomarker (e.g., natriuretic peptide or BNP) is the amount ofthe biomarker in a sample obtained prior to treatment. In someembodiments, the predetermined level of a biomarker (e.g., natriureticpeptide or BNP) is a normal reference level. In some embodiments, thepredetermined level for BNP is about 100 pg/ml or less in blood, serum,or plasma. In some embodiments, the normal reference level for BNP isabout 100 pg/ml or less in blood, serum, or plasma.

In any of the methods described herein, a biological sample is obtainedapproximately every week, every 2 weeks, every 3 weeks, or every 4weeks.

In some embodiments, if the BNP level in the sample is above apredetermined level for two consecutive samples, the subject isadministered a therapeutically effective amount of a cardioprotectivemedication such as an ACE inhibitor and/or a β-blocker. In someembodiments, if the BNP level in the biological sample is above 100pg/ml for two consecutive samples, the subject is administered atherapeutically effective amount of a cardioprotective medication suchas an ACE inhibitor and/or a β-blocker. In some embodiments of themethods described herein, if the BNP level in the biological sample isabove a predetermined level for any one sample, the subject isadministered a therapeutically effective amount of a cardioprotectivemedication such as an ACE inhibitor and/or a β-blocker. In someembodiments of the methods described herein, if the BNP level in thebiological sample is above 200 pg/ml for any one sample, the subject isadministered a therapeutically effective amount of a cardioprotectivemedication such as an ACE inhibitor and/or a β-blocker. In someembodiments of the methods described herein, if the BNP level in thebiological sample is above a predetermined level for any one sample, thesubject is administered a therapeutically effective amount of acardioprotective medication such as an ACE inhibitor and/or a β-blockerand the DLL4 antagonist is withheld. In some embodiments of the methodsdescribed herein, if the BNP level in the biological sample is above 300pg/ml for any one sample, the subject is administered a therapeuticallyeffective amount of a cardioprotective medication such as an ACEinhibitor and/or a β-blocker and the DLL4 antagonist is withheld. Insome embodiments, if the BNP level decreases to below 200 pg/ml afteradministration of an ACE inhibitor and/or a β-blocker, thenadministration of the DLL4 antagonist is resumed.

In some embodiments of any of the methods described herein, the subjectsare evaluated by LVEF monitoring. The LVEF represents the volumetricfraction of blood pumped out of the left ventricle of the heart witheach heart beat or cardiac cycle. Generally a normal range for LVEF is55-70%, and a significantly reduced ejection fraction typicallyindicates a cardiac dysfunction and/or heart failure. In someembodiments, cardiovascular dysfunction and/or cardiotoxicity isindicated if the LVEF is less than about 60%. In some embodiments,cardiovascular dysfunction and/or cardiotoxicity is indicated if theLVEF is less than about 55%. In some embodiments, cardiovasculardysfunction and/or cardiotoxicity is indicated if the LVEF is less thanabout 50%. In some embodiments, cardiovascular dysfunction and/orcardiotoxicity is indicated if there is a 10% or greater decline inLVEF. In some embodiments, cardiovascular dysfunction and/orcardiotoxicity is indicated if there is a 20% or greater decline inLVEF. In some embodiments, cardiovascular dysfunction and/orcardiotoxicity is indicated if there is a 20% or greater decline in LVEFto a value greater than 50%. In some embodiments, cardiovasculardysfunction and/or cardiotoxicity is indicated if there is a 10% orgreater decline in LVEF to a value less than 50%.

In some embodiments of any of the methods described herein, the subjectsare evaluated using a Doppler echocardiogram. Doppler echocardiographyis a method for detecting the direction and velocity of moving bloodwithin the heart and can be used to detect pulmonary hypertension.Pulmonary hypertension is high blood pressure that occurs in thearteries in the lungs. It is a different measurement altogether fromsystemic blood pressure and what is generally called “high bloodpressure” or “hypertension”. In some embodiments, cardiovasculardysfunction and/or cardiotoxicity is indicated if the subject has a peaktricuspid velocity (PTV) greater than 3.4 m/s on Doppler echocardiogram.In some embodiments, cardiovascular dysfunction and/or cardiotoxicity isindicated if the subject has a peak tricuspid velocity (PTV) greaterthan 3.4 m/s on Doppler echocardiogram that persists for more than 4weeks. In some embodiments, cardiovascular dysfunction and/orcardiotoxicity is indicated if the subject has a peak tricuspid velocity(PTV) greater than 3.4 m/s on Doppler echocardiogram that persists formore than 8 weeks.

The invention also provides methods of ameliorating cardiotoxicity in asubject administered a DLL4 antagonist, comprising: administering to thesubject a therapeutically effective amount of a cardioprotectivemedication such as an ACE inhibitor and/or a β-blocker. As describedherein, the cardiotoxicity is not systemic hypertension (i.e., highblood pressure).

The invention also provides methods of screening a subject for the riskof cardiovascular side effects and/or toxicity from treatment with aDLL4 antagonist, comprising: determining the level of a biomarker in asample from the subject, and comparing the level of the biomarker in thesample to a predetermined level of the biomarker, wherein if the levelof the biomarker in the sample is higher than the predetermined level ofthe biomarker then the subject is at risk for cardiovascular sideeffects and/or toxicity. In some embodiments, the methods of screening asubject for the risk of cardiovascular side effects and/or toxicity fromtreatment with a DLL4 antagonist comprise: obtaining a biological samplefrom the subject prior to treatment with the DLL4 antagonist,determining the level of a biomarker in the sample, and comparing thelevel of the biomarker in the sample to a predetermined level of thebiomarker, wherein if the level of the biomarker in the sample is higherthan the predetermined level of the biomarker then the subject is atrisk for cardiovascular side effects and/or toxicity. In someembodiments, the cardiovascular side effect and/or toxicity iscardiotoxicity. In some embodiments, the biomarker is a natriureticpeptide. In some embodiments, the natriuretic peptide is an atrialnatriuretic peptide (ANP)-type peptides, a brain natriuretic peptide(BNP)-type peptides, or variants thereof. ANP-type peptides includepre-proANP, proANP, NT-proANP, and ANP. BNP-type peptides includepre-proBNP, proBNP, NT-proBNP, and BNP. In some embodiments, thenatriuretic peptide is BNP. In some embodiments, the natriuretic peptideis NT-proBNP. In some embodiments, the natriuretic peptide is ANP. Insome embodiments, a method of screening a subject for the risk ofcardiotoxicity from treatment with a DLL4 antagonist comprises:obtaining a biological sample from the subject prior to treatment withthe DLL4 antagonist, determining the level of BNP in the sample, andcomparing the level of BNP in the sample to a predetermined level ofBNP, wherein if the level of BNP in the sample is higher than thepredetermined level of BNP then the subject is at risk forcardiotoxicity. In some embodiments, the predetermined level of BNP isabout 100 pg/ml. In some embodiments, if the subject is at risk forcardiotoxicity, the subject is administered a therapeutically effectiveamount of a cardioprotective medication such as an ACE inhibitor and/ora β-blocker prior to treatment with the DLL4 antagonist.

In some embodiments of the methods described herein, the ACE inhibitoris selected from the group consisting of: captopril, zofenopril,enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril,fosinopril, ceronapril, casokinins, lactokinins, teprotide, alacepril,cilazapril, delapril, imidapril, moexipril, rentiapril, spirapril,temocapril, moveltipril, and trandolapril.

In some embodiments of the methods described herein, the β-blocker isselected from the group consisting of: carvedilol, atenolol, metoprolol,nadolol, oxprenolol, pindolol, propranolol, timolol, acebutolol,bisoprolol, esmolol, labetalol, bucindolol, nebivolol, alprenolol;amosulalol, arotinolol, befunolol, betaxolol, bevantolot, bopindolol,bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrinehydrochloride, butofilolol, carazolol, carteolol, celiprolol, cetamolol,cloranololdilevalol, epanolol, indenolol, levobunolol, mepindolol,metipranolol, moprolol, nadoxolol, nipradilol, penbutolol, practolol,pronethalol, sotalol, sulfinalol, talinolol, tertatolol, tilisolol,toliprolol, and xibenolol. In some embodiments, the β-blocker iscarvedilol.

In any of the methods described herein, the DLL4 antagonist specificallybinds human DLL4. In some embodiments, the DLL4 antagonist is anantibody that specifically binds the extracellular domain of human DLL4.In some embodiments, the DLL4 antagonist specifically binds an epitopewithin amino acids 27-217 of the extracellular domain of human DLL4 (SEQID NO: 17). In some embodiments, the DLL4 antagonist binds an epitopecomprising amino acids 66-73 (QAVVSPGP, SEQ ID NO: 18) of human DLL4. Insome embodiments, the DLL4 antagonist binds an epitope comprising aminoacids 139-146 (LISKIAIQ, SEQ ID NO: 19) of human DLL4. In someembodiments, the DLL4 antagonist binds an epitope comprising amino acids66-73 (QAVVSPGP, SEQ ID NO: 18) and amino acids 139-146 (LISKIAIQ, SEQID NO: 19) of human DLL4. In some embodiments, the DLL4 antagonist bindshuman DLL4 with a dissociation constant (K_(D)) of about 10 nM to about0.1 nM.

In certain embodiments, the DLL4 antagonist is an anti-DLL4 antibody. Incertain embodiments, the DLL4 antagonist is an antibody comprising aheavy chain CDR1 comprising TAYYIH (SEQ ID NO: 1), a heavy chain CDR2comprising YISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS(SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:8). In certain embodiments, the DLL4 antagonist is an antibodycomprising a heavy chain variable region comprising the amino acids ofSEQ ID NO: 10. In certain embodiments, the DLL4 antagonist is anantibody which further comprises a light chain variable regioncomprising the amino acids of SEQ ID NO: 12. In certain embodiments, theDLL4 antagonist comprises the same heavy and light chain amino acidsequences as an antibody encoded by a plasmid deposited with ATCC havingdeposit no. PTA-8425 or PTA-8427. In certain embodiments, the DLL4antagonist comprises the heavy chain CDR amino acid sequences and thelight chain CDR amino acid sequences that are contained in the 21M18antibody produced by the hybridoma deposited with ATCC having depositno. PTA-8670. In certain embodiments, the DLL4 antagonist is encoded bythe plasmid having ATCC deposit no. PTA-8425 which was deposited withAmerican Type Culture Collection (ATCC), at 10801 University Boulevard,Manassas, Va., 20110, under the conditions of the Budapest Treaty on May10, 2007. In certain embodiments, the DLL4 antagonist is encoded by theplasmid having ATCC deposit no. PTA-8427 which was deposited withAmerican Type Culture Collection (ATCC), at 10801 University Boulevard,Manassas, Va., 20110, under the conditions of the Budapest Treaty on May10, 2007. In some embodiments, the DLL4 antagonist is the antibodyproduced by the hybridoma having ATCC deposit no. PTA-8670 which wasdeposited with the ATCC under the conditions of the Budapest Treaty onSep. 28, 2007. In some embodiments, the DLL4 antagonist is a humanizedversion of the antibody produced by the hybridoma having ATCC depositno. PTA-8670. In certain embodiments, the DLL4 antagonist competes forspecific binding to human DLL4 with an antibody encoded by the plasmiddeposited with ATCC having deposit no. PTA-8425 or PTA-8427.

In some embodiments, the subject has cancer. In some embodiments, thecancer is selected from the group consisting of: lung cancer, breastcancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer,gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroendocrine cancer, neuroblastoma, glioma, glioblastoma multiforme,cervical cancer, stomach cancer, bladder cancer, hepatoma, and head andneck cancer. In certain embodiments, the cancer is a hematologicalcancer, such as a lymphoma or leukemia. In certain embodiments, thecancer is non-small cell lung cancer (NSCLC). In certain embodiments,the cancer is ovarian cancer. In certain embodiments, the cancer ispancreatic cancer.

In some embodiments, the biological sample is a body fluid. In someembodiments, the biological sample is blood, plasma, serum, or urine. Insome embodiments, the biological sample is a venous whole bloodspecimen. In some embodiments, the biological sample is a venous wholeblood specimen using EDTA as an anticoagulant. In some embodiments, thebiological sample is a plasma specimen. In some embodiments, thebiological sample is a plasma specimen using EDTA as an anticoagulant.Samples of body fluids may be obtained by any method known in the art.In some embodiments, the biological sample is a frozen tissue sample oris fresh tissue sample.

Assays for measuring or determining the level of a natriuretic peptide(e.g., NT-proBNP or BNP) in a sample are known to those of skilled inthe art. For example, in some embodiments a fluorescent immunoassay thatquantitatively measures BNP levels in whole blood or plasma specimens isused. In some embodiments, the sample contains EDTA as an anticoagulant.In some embodiments, the test is performed at the bedside. In someembodiments, a sample is placed in a test device and the sample moves bycapillary action into a reaction chamber containing murine fluorescentantibodies to BNP. The reaction mixture then flows through an elutioncolumn. Analyte and fluorescent antibody-BNP conjugates are captured indiscrete zones along the column. Bound fluorescent material representsthe serum BNP concentration. After about 15 minutes, the test device isplaced in an immunofluorescence reader and the BNP concentration isdetermined. BNP levels less than or equal to 100 pg/ml are consideredrepresentative of normal values in patients without congestive heartfailure (CHF) by those skilled in the art. BNP levels above 100 pg/ml to300 ug/ml are suggestive of heart failure, BNP levels above 300 pg/mlindicate mild heart failure, BNP levels above 600 pg/ml indicatemoderate heart failure, and BNP levels above 900 pg/ml indicate severeheart failure.

In some embodiments, the DLL4 antagonist is administered as an initialdose of about 2.5 mg/kg. For example, antibody OMP-21M18 is diluted with5% dextrose in water (USP) to a total volume of 250 mL. The OMP-21M18 isdelivered through a 0.22-micron filter over 30 minutes as an intravenousinfusion. In some embodiments, subsequent doses are administered in asimilar manner.

In another aspect of the invention, the methods described herein mayfurther comprise administering one or more additional therapeuticagents. An additional therapeutic agent can be administered prior to,concurrently with, and/or subsequently to, administration of the DLL4antagonist. Pharmaceutical compositions comprising a DLL4 antagonist andan additional therapeutic agent(s) are also provided. In someembodiments, the one or more additional therapeutic agents comprise 1,2, 3, or more additional therapeutic agents.

Combination therapy with at least two therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing side effects and/or toxicities. Combination therapy mayincrease the therapeutic index of one or both of the therapeutic agents.Combination therapy may decrease the likelihood that resistant cancercells will develop. In some embodiments, combination therapy comprises atherapeutic agent that primarily affects (e.g., inhibits or kills)non-tumorigenic cells and a therapeutic agent that primarily affects(e.g., inhibits or kills) tumorigenic CSCs.

It will be appreciated that the combination of a DLL4 antagonist and anadditional therapeutic agent may be administered in any order orconcurrently. In some embodiments, the DLL4 antagonist is administeredto subjects that have previously undergone treatment with a secondtherapeutic agent. In certain other embodiments, the DLL4 antagonist anda second therapeutic agent is administered substantially simultaneouslyor concurrently. For example, a subject may be given a DLL4 antagonist(e.g., an antibody) while undergoing a course of treatment with a secondtherapeutic agent (e.g., chemotherapy). In certain embodiments, a DLL4antagonist is administered within 1 year of the treatment with a secondtherapeutic agent. In certain alternative embodiments, a DLL4 antagonistis administered within 10, 8, 6, 4, or 2 months of any treatment with asecond therapeutic agent. In certain other embodiments, a DLL4antagonist is administered within 4, 3, 2, or 1 weeks of any treatmentwith a second therapeutic agent. In some embodiments, a DLL4 antagonistis administered within 5, 4, 3, 2, or 1 days of any treatment with asecond therapeutic agent. It will further be appreciated that the two(or more) agents or treatments may be administered to the subject withina matter of hours or minutes (i.e., substantially simultaneously).

As is known to those of skill in the art, administration of anytherapeutic agent may lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, drug therapy must be discontinued, and other agentsmay be tried. However, many agents in the same therapeutic class oftendisplay similar side effects and/or toxicities, meaning that the subjecteither has to stop therapy, or if possible, suffer from the unpleasantside effects associated with the therapeutic agent.

Side effects from therapeutic agents may include, but are not limitedto, hives, skin rashes, itching, nausea, vomiting, decreased appetite,diarrhea, chills, fever, fatigue, muscle aches and pain, headaches, lowblood pressure, high blood pressure, hypokalemia, low blood counts,bleeding, and cardiac problems.

Thus, in some embodiments, the methods described herein include using anintermittent dosing regimen, which may reduce side effects and/ortoxicities associated with administration of a DLL4 antagonist. As usedherein, “intermittent dosing” refers to a dosing regimen using a dosinginterval of more than once a week, e.g., dosing once every 2 weeks, onceevery 3 weeks, once every 4 weeks, etc. In some embodiments, a methodfor treating a subject comprises administering to the subject aneffective dose of a DLL4 antagonist (e.g., an anti-DLL4 antibody)according to an intermittent dosing regimen. In some embodiments, themethod comprises administering to the subject an effective dose of aDLL4 antagonist (e.g., an anti-DLL4 antibody) according to anintermittent dosing regimen, and increasing the therapeutic index of theDLL4 antagonist. In some embodiments, the intermittent dosing regimencomprises administering an initial dose of a DLL4 antagonist to thesubject, and administering subsequent doses of the DLL4 antagonist aboutonce every 2 weeks. In some embodiments, the intermittent dosing regimencomprises administering an initial dose of a DLL4 antagonist to thesubject, and administering subsequent doses of the DLL4 antagonist aboutonce every 3 weeks. In some embodiments, the intermittent dosing regimencomprises administering an initial dose of a DLL4 antagonist to thesubject, and administering subsequent doses of the DLL4 antagonist aboutonce every 4 weeks.

In some embodiments, the subsequent doses in an intermittent dosingregimen are about the same amount or less than the initial dose. Inother embodiments, the subsequent doses are a greater amount than theinitial dose. As is known by those of skill in the art, doses used willvary depending on the clinical goals to be achieved. In someembodiments, the initial dose is about 0.25 mg/kg to about 20 mg/kg. Insome embodiments, the initial dose is about 0.25, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In certainembodiments, the initial dose is about 0.5 mg/kg. In certainembodiments, the initial dose is about 1 mg/kg. In certain embodiments,the initial dose is about 2.5 mg/kg. In certain embodiments, the initialdose is about 5 mg/kg. In certain embodiments, the initial dose is about7.5 mg/kg. In certain embodiments, the initial dose is about 10 mg/kg.In certain embodiments, the initial dose is about 12.5 mg/kg. In certainembodiments, the initial dose is about 15 mg/kg. In certain embodiments,the initial dose is about 20 mg/kg. In some embodiments, the subsequentdoses are about 0.25 mg/kg to about 15 mg/kg. In certain embodiments,the subsequent doses are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 mg/kg. In certain embodiments, the subsequent doses areabout 0.5 mg/kg. In certain embodiments, the subsequent doses are about1 mg/kg. In certain embodiments, the subsequent doses are about 2.5mg/kg. In certain embodiments, the subsequent doses are about 5 mg/kg.In some embodiments, the subsequent doses are about 7.5 mg/kg. In someembodiments, the subsequent doses are about 10 mg/kg. In someembodiments, the subsequent doses are about 12.5 mg/kg.

In some embodiments, the intermittent dosing regimen comprises: (a)administering to the subject an initial dose of a DLL4 antagonist ofabout 2.5 mg/kg and (b) administering subsequent doses of about 2.5mg/kg once every 2 weeks. In some embodiments, the intermittent dosingregimen comprises: (a) administering to the subject an initial dose of aDLL4 antagonist of about 5 mg/kg and (b) administering subsequent dosesof about 5 mg/kg once every 2 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the subjectan initial dose of a DLL4 antagonist of about 2.5 mg/kg and (b)administering subsequent doses of about 2.5 mg/kg once every 3 weeks. Insome embodiments, the intermittent dosing regimen comprises: (a)administering to the subject an initial dose of a DLL4 antagonist ofabout 5 mg/kg and (b) administering subsequent doses of about 5 mg/kgonce every 3 weeks. In some embodiments, the intermittent dosing regimencomprises: (a) administering to the subject an initial dose of a DLL4antagonist of about 2.5 mg/kg and (b) administering subsequent doses ofabout 2.5 mg/kg once every 4 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the subjectan initial dose of a DLL4 antagonist of about 5 mg/kg and (b)administering subsequent doses of about 5 mg/kg once every 4 weeks. Incertain embodiments, the initial dose and the maintenance doses aredifferent, for example, the initial dose is about 5 mg/kg and thesubsequent doses are about 2.5 mg/kg. In certain embodiments, anintermittent dosing regimen may comprise a loading dose, for example,the initial dose is about 20 mg/kg and the subsequent doses are about2.5 mg/kg or about 5 mg/kg administered once every 2 weeks, once every 3weeks, or once every 4 weeks.

Another aspect of the present invention is directed to methods forreducing toxicity of a DLL4 antagonist in a human subject comprisesadministering to the subject the DLL4 antagonist using an intermittentdosing regimen. Another aspect of the present invention is directed tomethods for reducing side effects of a DLL4 antagonist in a humansubject comprises administering to the subject the DLL4 antagonist usingan intermittent dosing regimen. Another aspect of the present inventionis directed to methods for increasing the therapeutic index of a DLL4antagonist in a human subject comprises administering to the subject theDLL4 antagonist using an intermittent dosing regimen.

The choice of delivery method for the initial and subsequent doses ismade according to the ability of the subject to tolerate introduction ofthe DLL4 antagonist into the body. Thus, in any of the aspects and/orembodiments described herein, the administration of the DLL4 antagonistmay be by intravenous injection or intravenously. In some embodiments,the administration is by intravenous infusion. In any of the aspectsand/or embodiments described herein, the administration of the DLL4antagonist may be by a non-intravenous route.

Therapeutic agents that may be administered in combination with the DLL4antagonist include chemotherapeutic agents. Thus, in some embodiments,the method or treatment involves the administration of a DLL4 antagonistof the present invention in combination with a chemotherapeutic agent orcocktail of multiple different chemotherapeutic agents. Treatment with aDLL4 antagonist (e.g., an antibody) can occur prior to, concurrentlywith, or subsequent to administration of chemotherapies. Combinedadministration can include co-administration, either in a singlepharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in The Chemotherapy Source Book, 4th Edition, 2008, M. C.Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the instant invention include, but arenot limited to, alkylating agents such as thiotepa and cyclophosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel(TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including, for example, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, theadditional therapeutic agent is cisplatin. In certain embodiments, theadditional therapeutic agent is carboplatin. In certain embodiments, theadditional therapeutic agent is paclitaxel. In certain embodiments,where the chemotherapeutic agent administered in combination with a DLL4antagonist is carboplatin, the cancer or tumor being treated is lungcancer or a lung tumor.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapeutic agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese. In certain embodiments, the additional therapeutic agent isirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the additional therapeutic agent isgemcitabine. In some embodiments, the additional therapeutic agent ispemetrexed. In certain embodiments, where the chemotherapeutic agentadministered in combination with a DLL4 antagonist is gemcitabine, thecancer or tumor being treated is pancreatic cancer or a pancreatictumor. In certain embodiments, where the chemotherapeutic agentadministered in combination with a DLL4 antagonist is pemetrexed, thecancer or tumor being treated is lung cancer or a lung tumor. In someembodiments, the DLL4 antagonist is administered in combination withpemetrexed and carboplatin.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain embodiments, the agentis paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel(ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor ofa mitotic kinase such as Aurora A or Plk 1. In certain embodiments,where the chemotherapeutic agent administered in combination with a DLL4antagonist is an anti-mitotic agent, the cancer or tumor being treatedis breast cancer or a breast tumor.

In some embodiments, an additional therapeutic agent comprises an agentsuch as a small molecule. For example, treatment can involve thecombined administration of a DLL4 antagonist (e.g. an antibody) of thepresent invention with a small molecule that acts as an inhibitoragainst additional tumor-associated proteins including, but not limitedto, EGFR, ErbB2, HER2, and/or VEGF. In certain embodiments, theadditional therapeutic agent is a small molecule that inhibits a cancerstem cell pathway. In some embodiments, the additional therapeutic agentis a small molecule inhibitor of the Notch pathway. In some embodiments,the additional therapeutic agent is a small molecule inhibitor of theWnt pathway. In some embodiments, the additional therapeutic agent is asmall molecule inhibitor of the BMP pathway. In some embodiments, theadditional therapeutic agent is a small molecule that inhibits β-cateninsignaling.

In some embodiments, an additional therapeutic agent comprises abiological molecule, such as an antibody. For example, treatment caninvolve the combined administration of a DLL4 antagonist (e.g. anantibody) of the present invention with other antibodies againstadditional tumor-associated proteins including, but not limited to,antibodies that bind EGFR, ErbB2, HER2, and/or VEGF. In certainembodiments, the additional therapeutic agent is an antibody that is ananti-cancer stem cell marker antibody. In some embodiments, theadditional therapeutic agent is an antibody that binds a component ofthe Notch pathway. In some embodiments, the additional therapeutic agentis an antibody that binds a component of the Wnt pathway. In certainembodiments, the additional therapeutic agent is an antibody thatinhibits a cancer stem cell pathway. In some embodiments, the additionaltherapeutic agent is an antibody inhibitor of the Notch pathway. In someembodiments, the additional therapeutic agent is an antibody inhibitorof the Wnt pathway. In some embodiments, the additional therapeuticagent is an antibody inhibitor of the BMP pathway. In some embodiments,the additional therapeutic agent is an antibody that inhibits β-cateninsignaling. In certain embodiments, the additional therapeutic agent isan antibody that is an angiogenesis inhibitor or modulator (e.g., ananti-VEGF or VEGF receptor antibody). In certain embodiments, theadditional therapeutic agent is bevacizumab (AVASTIN), trastuzumab(HERCEPTIN), panitumumab (VECTIBIX), or cetuximab (ERBITUX). Combinedadministration can include co-administration, either in a singlepharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

Furthermore, treatment with a DLL4 antagonist described herein caninclude combination treatment with other biologic molecules, such as oneor more cytokines (e.g., lymphokines, interleukins, tumor necrosisfactors, and/or growth factors) or can be accompanied by surgicalremoval of tumors, cancer cells, or any other therapy deemed necessaryby a treating physician.

In certain embodiments, the treatment involves the administration of aDLL4 antagonist (e.g. an antibody) of the present invention incombination with radiation therapy. Treatment with a DLL4 antagonist canoccur prior to, concurrently with, or subsequent to administration ofradiation therapy. Dosing schedules for such radiation therapy can bedetermined by the skilled medical practitioner.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe the useof a DLL4 antagonist for treatment of cancer. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of thepresent disclosure.

EXAMPLES Example 1

Phase 1 Study of OMP-21M18 in Subjects with Previously Treated SolidTumors

The study was an open-label Phase 1 dose-escalation study of OMP-21M18in subjects with advanced solid tumors. The primary objective of thestudy was to determine the maximum tolerated dose of OMP-21M18. Thesecondary objectives were to determine the safety, the rate ofimmunogenicity, the preliminary efficacy, and the pharmacokinetics ofOMP-21M18.

The subjects in the initial portion of the trial were treated at doselevels of 0.5 (n=3), 1.0 (n=3), 2.5 (n=6), and 5.0 mg/kg (n=3) once aweek for nine doses and then every other week; 2.5 (n=6), 5 (n=6) and 10mg/kg (n=12) once every other week; and 10 mg/kg on Days 0, 7, and 14 asa loading dose and then once every other week. In the expansion portionof the study, 15 additional subjects were treated with 10 mg/kg onceevery other week. Cohorts of 3 subjects were treated and evaluated fordose-limiting toxicities (DLTs) through Day 28. If 0 of 3 subjects had aDLT, escalation to the next dose cohort occurred. If 1 of 3 subjects hada DLT, 3 additional subjects were treated and escalation to the nextcohort occurred if less than 2 of 6 subjects experienced a DLT. Subjectscontinued on treatment until Day 56, when an assessment of the tumorrespond was performed.

The maximum tolerated dose was not reached at a dose of 10 mg/kg everyother week, however several subjects treated with 10 mg/kg every otherweek for longer than 100 days showed signs of cardiotoxicity thatexceeded the level of acceptable chronic toxicity and enrollment in thestudy was stopped early.

During the conduct of the Phase 1 study, cardiotoxicity was identifiedas a potential toxicity in an ongoing monkey study. Based on the initialPhase 1 study results and the monkey study results, the Phase 1 studyprotocol was amended to include monitoring for cardiac dysfunctionand/or cardiotoxicity with BNP measurements and echocardiograms. Asdiscussed herein, BNP (or NT-proBNP) levels in blood/serum samples maybe used to detect cardiac dysfunction and/or heart failure. Increases inBNP levels to greater than 400 pg/ml or NT-proBNP levels to greater than800 pg/ml considered to be possibly related to OMP-21M18 treatment wereobserved in six patients who received 10 mg/kg once every other week.Five of the patients who received 10 mg/kg once every other week hadLVEF declines as evaluated by echocardiograms and four of these patientsdeveloped congestive heart failure. OMP-21M18 treatment was discontinuedfor all four patients and they were administered medications for heartfailure. The symptoms of heart failure subsequently diminished in allsubjects. No instances of significant increases in BNP levels or othersigns of cardiotoxicity were observed in the lower dose cohorts, exceptfor one patient who received 2.5 mg/kg once every other week anddeveloped pulmonary hypertension.

Example 2

Phase 1b Study of OMP-21M18 in Combination with Carboplatin andPemetrexed in Subjects with NSCLC

The study is a Phase 1b dose-escalation study of OMP-21M18 pluscarboplatin and pemetrexed (carbo/PEM) in subjects with unresectable,locally advanced, recurrent, or metastatic non-squamous non-small celllung cancer (NSCLC). Subjects had not received prior chemotherapy forthe cancer. The primary objective of the study was to determine themaximum tolerated dose of OMP-21M18 plus carbo/PEM in subjects withNSCLC. The secondary objectives were to determine the safety, the rateof immunogenicity, the preliminary efficacy, and the pharmacokinetics ofOMP-21M18 in combination with carbo/PEM as a first line treatment insubjects with NSCLC.

Carboplatin (6 mg/ml×min×[creatinine clearance (ml/min)+25]) andpemetrexed (500 mg/m²) were administered once every 21 days for a totalof 6 cycles (or for less than 6 full cycles if toxicity necessitatesreducing or holding a dose or terminating treatment). Patients withstable disease or a response at the end of the 6 cycles continued toreceive OMP-21M18 once every 3 weeks as maintenance therapy. OMP-21M18was supplied at a concentration of 10 mg/ml in a 25-ml single-use glassvial filled to 20 ml to deliver a total of 200 mg per vial. OMP-21M18was administered by intravenous (IV) infusion over 30 minutes once every21 days (on the same day as the scheduled carbo/PEM administration)until disease progression.

Patient demographics are summarized in Table 1.

TABLE 1 Dose Cohort (mg/kg Q3W) 5* 2.5 5 Total No. of patients 6 6 8 20Median age (years) 66.5 59.5 65.0 64.0 Male/Female 2/4 2/4 2/6 6/14Prior Surgery 0 0 1 1 Prior Neoadjuvant/ 0 0 0 0 Adjuvant Therapy PriorRadiotherapy 2 1 5 8 *Prior to risk mitigation

The first cohort of 6 patients was administered OMP-21M18 at a dosage of5 mg/kg once every 3 weeks. Treatment in this cohort was paused due toemerging evidence of cardiotoxicity secondary to administration ofOMP-21M18 in other ongoing studies (see Example 1). The Phase 1bprotocol was amended to include a risk mitigation plan to enhance thetherapeutic index of OMP-21M18 and manage tolerability. The secondcohort of six patients was administered OMP-21M18 at a dosage of 2.5mg/kg once every 3 weeks and the third cohort of eight patients wasadministered OMP-21M18 at a dosage of 5 mg/kg once every 3 weeks. Aspart of the safety evaluation and risk mitigation, subjects weremonitored for B-type natriuretic peptide (BNP) levels in blood/serumsamples every 14 days using an Alere Triage BNP test and device.Patients were administered a cardioprotective ACE inhibitor and/orcarvedilol if their BNP levels were greater than or equal to 100 pg/mlin two consecutive samples or greater than or equal to 200 pg/ml in onesample, while still receiving OMP-21M18. Patients were administered anACE inhibitor or carvedilol if their BNP level was greater than or equalto 300 pg/ml in any one sample and treatment with OMP-21M18 waswithheld. Treatment with OMP-21M18 was restarted when BNP levelsdecreased below 300 pg/ml. If a subject's BNP level was greater than 400pg/ml in any one sample, treatment with OMP-21M18 was discontinued. Inaddition, subjects had Doppler echocardiograms every 28 days to assesscardiac function and monitor left ventricular ejection fraction (LVEF)and peak tricuspid velocity (PTV). Baseline BNP and echocardiogramreadings were taken just prior to administration of the first dosage.

During the study, subjects were assessed for adverse events from thetime of enrollment through 30 days after the last dose of OMP-21M18.Adverse events were assessed using the National Cancer Institute (NCI)Common Terminology Criteria for Adverse Events (CTCAE), version 4.02.

In the first cohort (OMP-21M18 5 mg/kg once every 3 weeks), two patientshad a partial response, two patients had stable disease, and twopatients were not evaluable. Two patients had no progressive disease forgreater than 20 months. One of the six patients had an increase in BNP,with no cardiac impairment observed on echocardiograms and no evidenceof congestive heart failure. As noted above, initial treatment of thiscohort predated implementation of the risk mitigation strategy forcohorts 2 and 3.

In the second cohort (OMP-21M18 2.5 mg/kg once every 3 weeks), fourpatients had a partial response and two patients had stable disease,with the response lasting from 112 to 225 days. Three of six patientshad an increase in their BNP levels and two of those patients met thecriteria for administration of a cardioprotective ACE inhibitor and/orcarvedilol. The third patient only had one sample with a BNP level above100 pg/ml, and was not treated with an ACE inhibitor and/or carvedilol.There was no cardiac impairment in these patients observed onechocardiograms and no evidence of congestive heart failure.

In the third cohort (OMP-21M18 5 mg/kg once every 3 weeks), two patientshad a partial response, four patients had stable disease, one patienthad progressive disease due to a new lesion, and one patient was notevaluable (this cohort is ongoing). Four of eight patients had anincrease in their BNP levels and three patients were treated with acardioprotective ACE inhibitor and/or carvedilol. The fourth patientonly had one sample with a BNP level at or above 100 pg/ml, and was nottreated with an ACE inhibitor and/or carvedilol. There was no cardiacimpairment in these patients observed on echocardiograms and no evidenceof congestive heart failure. These results are summarized in FIG. 1.

In regard to efficacy as measured by the percentage change in targettumor lesions including cohorts 1, 2 and 3, the response of evaluablepatients was: 8/17 (47%) patients had a partial response, 8/17 (47%)patients had stable disease, and 1/17 (6%) patients had progressivedisease (FIG. 2).

In regard to safety in cohorts 2 and 3 with risk mitigation, fivepatients were started on a cardioprotective ACE inhibitor and/orcarvedilol. Three of the patients continued treatment with OMP-21M18 andtheir BNP levels were controlled, while one patient was taken offOMP-21M18 treatment due to an increased BNP level above 400 pg/ml. Noneof the patients had a decline in LVEF or evidence of congestive heartfailure. Other aspects of the safety profile were similar to what hasbeen observed with standard carboplatin/pemetrexed chemotherapytreatment. In addition, in a preliminary analysis of patient samples(n=10), pemetrexed and carboplatin did not appear to influence thepharmacokinetics of OMP-21M18.

Example 3

Phase 1b Study of OMP-21M18 in Combination with Gemcitabine in Subjectswith Pancreatic Cancer

The study is a Phase 1b dose-escalation study of OMP-21M18 plusgemcitabine in subjects with locally advanced or metastatic pancreaticcancer. Subjects had not received prior chemotherapy for the cancer. Theprimary objective of the study was to determine the maximum tolerateddose of OMP-21M18 plus gemcitabine in subjects with pancreatic cancer.The secondary objectives were to determine the safety, the rate ofimmunogenicity, the preliminary efficacy, and the pharmacokinetics ofOMP-21M18 in combination with gemcitabine as a first line treatment insubjects with pancreatic.

Gemcitabine (1000 mg/m2) was administered once every week for up to 7weeks (or until toxicity necessitates reducing or holding a dose),followed by a week of rest from treatment. Subsequent cycles consistedof once weekly infusions for 3 consecutive weeks out of every 4 weeks.OMP-21M18 was supplied at a concentration of 10 mg/ml in a 25-mlsingle-use glass vial filled to 20 ml to deliver a total of 200 mg pervial. OMP-21M18 was administered by intravenous (IV) infusion over 30minutes once every 14 days or once every 4 weeks until diseaseprogression.

The first cohort of eight patients was administered OMP-21M18 at adosage of 2.5 mg/kg once every 2 weeks. Treatment in this cohort waspaused due to emerging evidence of cardiotoxicity secondary toadministration of OMP-21M18 in other ongoing studies (see Example 1).The protocol was amended to include a risk mitigation plan to enhancethe therapeutic index of OMP-21M18 and manage tolerability. The secondcohort of eight patients was administered OMP-21M18 at a dosage of 2.5mg/kg once every 4 weeks and the third cohort of eight patients will beadministered OMP-21M18 at a dosage of 5 mg/kg once every 4 weeks. Aspart of the safety evaluation and risk mitigation, subjects weremonitored for B-type natriuretic peptide (BNP) levels in blood samplesevery 14 days. Patients were administered a cardioprotective ACEinhibitor or carvedilol if their BNP levels were greater than or equalto 100 pg/ml in two consecutive samples or greater than or equal to 200pg/ml in one sample, while still receiving OMP-21M18. Patients wereadministered an ACE inhibitor or carvedilol if their BNP level wasgreater than or equal to 300 pg/ml in any one sample and treatment withOMP-21M18 was withheld. Treatment with OMP-21M18 was restarted when BNPlevels decreased below 300 pg/ml. If a subject's BNP level was greaterthan 400 pg/ml in any one sample, treatment with OMP-21M18 wasdiscontinued. In addition, subjects had Doppler echocardiograms every 28days to assess cardiac function and monitor left ventricular ejectionfraction (LVEF) and peak tricuspid velocity (PTV). Baseline BNP andechocardiogram readings were taken just prior to administration of thefirst dosage.

During the study, subjects were assessed for adverse events from thetime of enrollment through 30 days after the last dose of OMP-21M18.Adverse events were assessed using the National Cancer Institute (NCI)Common Terminology Criteria for Adverse Events (CTCAE), version 4.02.

In the first cohort (OMP-21M18 2.5 mg/kg once every 2 weeks), onepatient had a partial response, four patients had stable disease, andthree patients were not evaluable. Six of eight patients had noprogressive disease for 22 to 147 days, before study was put on hold.Six of the eight patients had an increase in their BNP, with no cardiacimpairment observed on echocardiograms and no evidence of congestiveheart failure. As noted above, initial treatment of this cohort predatedimplementation of the risk mitigation strategy for cohorts 2 and 3.

In the second cohort (OMP-21M18 2.5 mg/kg once every 4 weeks), onepatient had a partial response, two patients had stable disease, threepatients had progressive disease, and two patients were not evaluable.This cohort is ongoing. Four of eight patients had an increase in theirBNP levels and one of those patients met the criteria for administrationof an ACE inhibitor and/or carvedilol. There was no cardiac impairmentin these patients observed on echocardiograms and no evidence ofcongestive heart failure.

In regard to efficacy as measured by the percentage change in targettumor lesions including cohorts 1, and 2, the response for evaluablepatients was: 2/11 (18%) patients had a partial response, 6/11 (54%)patients had stable disease, and 3/11 (27%) patients had progressivedisease (FIG. 3). In regard to safety in cohort 2 with risk mitigation,1 patient was started on a cardioprotective ACE inhibitor and/orcarvedilol while continuing treatment with OMP-21M18 and their BNPlevels were controlled. In all patients, there was no decline seen inLVEF or evidence of congestive heart failure. Other aspects of thesafety profile were similar to what has been observed with standardgemcitabine chemotherapy treatment.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference.

SEQUENCES  21M18 Heavy chain CDR1 (SEQ ID NO: 1)  TAYYIH 21M18-H2 Heavy chain CDR2 (SEQ ID NO: 2)  YISCYNGATNYNQKFKG 21M18-H7 Heavy chain CDR2 (SEQ ID NO: 3)  YISSYNGATNYNQKFKG 21M18-H9 Heavy chain CDR2 (SEQ ID NO: 4)  YISVYNGATNYNQKFKG 21M18 Heavy chain CDR3 (SEQ ID NO: 5)  RDYDYDVGMDY 21M18 Light chain CDR1 (SEQ ID NO: 6)  RASESVDNYGISFMK 21M18 Light chain CDR2 (SEQ ID NO: 7)  AASNQGS 21M18 Light chain CDR3 (SEQ ID NO: 8)  QQSKEVPWT FGG 21M18-H2 Heavy chain variable region (SEQ ID NO: 9) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISCYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21M18-H7 Heavy chain variable region (SEQ ID NO: 10) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21M18-H9 Heavy chain variable region (SEQ ID NO: 11) QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISVYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21M18 Light chain variable region (SEQ ID NO: 12) DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVE1KHuman DLL4 extracellular domain with putative signal sequenceunderlined (SEQ ID NO: 13) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSILSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFIGTYCELHVSDCARNPCAHGGICHDLENGLMCICPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGHuman DLL4 N-terminal region with putative signal sequence underlined (SEQ ID NO: 14) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNHuman DLL4 DSL Region (SEQ ID NO: 15) WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTG  EYC Human DLL4 amino acids 1-217 with putative signal sequenceunderlined (SEQ ID NO: 16) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCHuman DLL4 amino acids 27-217 (SEQ ID NO: 17) SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYC  Human DLL4 amino acids 66-73 (SEQ ID NO: 18)  QAVVSPGP Human DLL4 amino acids 139-146 (SEQ ID NO: 19)  LISKIAIQ 

1-16. (canceled)
 17. A method of detecting the development of acardiovascular side effect and/or toxicity in a subject receivingtreatment with a DLL4 antagonist, comprising: (a) determining the levelof a natriuretic peptide in a sample from the subject; and (b) comparingthe level of the natriuretic peptide in the sample to a predeterminedlevel of the natriuretic peptide; wherein an increase in the level ofthe natriuretic peptide indicates development of a cardiovascular sideeffect and/or toxicity. 18-19. (canceled)
 20. The method of claim 17,wherein the sample is blood, serum, or plasma.
 21. The method of claim17, wherein the cardiovascular side effect and/or toxicity iscardiotoxicity.
 22. The method of claim 17, wherein the predeterminedlevel of the natriuretic peptide is the amount of natriuretic peptide ina sample obtained at an earlier date.
 23. The method of claim 17,wherein the predetermined level of the natriuretic peptide is the amountof natriuretic peptide in a sample obtained prior to treatment.
 24. Themethod of claim 17, wherein the predetermined level of the natriureticpeptide is a normal reference level.
 25. The method of claim 24, whereinthe normal reference level for BNP is about 100 pg/ml or less in blood,serum, or plasma.
 26. The method of claim 17, wherein a sample isobtained approximately every 2 weeks.
 27. The method of claim 26,wherein if the natriuretic peptide level is above a predetermined levelfor two consecutive samples, the subject is administered atherapeutically effective amount of an ACE inhibitor and/or a β-blocker.28. The method of claim 17, wherein if the natriuretic peptide level isabove a predetermined level for any one sample, the subject isadministered a therapeutically effective amount of an ACE inhibitorand/or a β-blocker.
 29. The method of claim 17, wherein if thenatriuretic peptide level is above a predetermined level for any onesample, the subject is administered a therapeutically effective amountof an ACE inhibitor and/or a β-blocker and the DLL4 antagonist iswithheld.
 30. The method of claim 17, wherein the natriuretic peptide isB-type natriuretic peptide (BNP).
 31. The method of claim 30, whereinthe predetermined level of BNP is about 100 pg/ml in blood, serum, orplasma.
 32. The method of claim 30, wherein the predetermined level ofBNP is about 200 pg/ml in blood, serum, or plasma.
 33. The method ofclaim 30, wherein the predetermined level of BNP is about 300 pg/ml inblood, serum, or plasma.
 34. The method of claim 29, wherein if the BNPlevel decreases to below about 200 pg/ml after administration of the ACEinhibitor and/or a β-blocker, then administration of the DLL4 antagonistis resumed.
 35. The method of claim 28, wherein if the BNP leveldecreases after administration of the ACE inhibitor and/or a β-blocker,then administration of the DLL4 antagonist is resumed. 36-45. (canceled)46. The method of claim 17, wherein the DLL4 antagonist is an antibodythat specifically binds human DLL4.
 47. The method of claim 17, whereinthe DLL4 antagonist is an antibody comprising: a heavy chain CDR1comprising TAYYIH (SEQ ID NO: 1), a heavy chain CDR2 comprisingYISSYNGATNYNQKFKG (SEQ ID NO:3), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:5), and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:6), a light chain CDR2 comprising AASNQGS(SEQ ID NO:7), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:8).
 48. The method of claim 17, wherein the DLL4 antagonist is anantibody comprising: a heavy chain variable region comprising SEQ IDNO:10 and a light chain variable region comprising SEQ ID NO:12.
 49. Themethod of claim 17, wherein the DLL4 antagonist is antibody OMP-21M18.50. The method of claim 17, wherein the DLL4 antagonist is ananti-DLL4/anti-VEGF bispecific antibody.
 51. The method of claim 28,wherein the ACE inhibitor is selected from the group consisting of:captopril, zofenopril, enalapril, ramipril, quinapril, perindopril,lisinopril, benazepril, fosinopril, ceronapril, casokinins, lactokinins,teprotide, alacepril, cilazapril, delapril, imidapril, moexipril,rentiapril, spirapril, temocapril, moveltipril and trandolapril.
 52. Themethod of claim 28, wherein the β-blocker is selected from the groupconsisting of: carvedilol, atenolol, metoprolol, nadolol, oxprenolol,pindolol, propranolol, timolol, acebutolol, bisoprolol, esmolol,labetalol, bucindolol, nebivolol, alprenolol; amosulalol, arotinolol,befunolol, betaxolol, bevantolot, bopindolol, bucumolol, bufetolol,bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol,carazolol, carteolol, celiprolol, cetamolol, cloranololdilevalol,epanolol, indenolol, levobunolol, mepindolol, metipranolol, moprolol,nadoxolol, nipradilol, penbutolol, practolol, pronethalol, sotalol,sulfinalol, talinolol, tertatolol, tilisolol, toliprolol, and xibenolol.53. The method of claim 28, wherein the β-blocker is carvedilol.
 54. Themethod of claim 17, wherein the subject has cancer.
 55. The method ofclaim 54, wherein the cancer is selected from the group consisting of:lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma,pancreatic cancer, gastrointestinal cancer, renal cancer, ovariancancer, liver cancer, endometrial cancer, kidney cancer, prostatecancer, thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme,cervical cancer, stomach cancer, bladder cancer, hepatoma, and head andneck cancer.
 56. The method of claim 54, wherein the subject is treatedwith the DLL4 antagonist in combination with one or more additionalanti-cancer agents.
 57. The method of claim 17, wherein thecardiovascular side effect and/or toxicity is related to the DLL4antagonist.
 58. The method of claim 17, wherein the cardiovascular sideeffect and/or toxicity is left ventricular dysfunction or congestiveheart failure.
 59. The method of claim 50, wherein theanti-DLL4/anti-VEGF bispecific antibody is selected from the groupconsisting of 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, and219R45-MB-21R83.
 60. The method of claim 59, wherein the antibody is219R45-MB-21R83.